Human Elongase Genes, Uses Thereof, and Compounds for Modulating Same

ABSTRACT

The present invention relates to elongase genes, their polypeptides and their control regions, and the use of such genes, polypeptides and control regions in determining compositions for use in the treatment of disease. The identified compositions regulate the expression of the elongase genes or modulate the activity of their protein products. The nucleotide and amino acid sequences are taught for ELG4, ELG6 and ELG7. The control sequences and function are taught for ELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a divisional of co-pending U.S. patent applicationSer. No. 10/433,238, filed Nov. 20, 2003, which is the National Stage ofInternational Application No. PCT/CA01/01705 filed Nov. 29, 2001, whichclaims priority to U.S. Provisional Application No. 60/253,728 (nowexpired), filed Nov. 29, 2000. All of the above priority documents areincorporated herewith by reference in their entirety.

FIELD OF THE INVENTION

This invention relates to the identification of compounds that modulatethe activity of fatty acid elongase enzymes involved in lipid metabolismand/or effectively regulate the level of expression of the elongasegenes, and to compounds so identified.

BACKGROUND OF THE INVENTION

Polyunsaturated fatty acids (PUFAs) are major components of lipidcompounds and complexes, such as phospholipids and lipoproteins, whichprovide a number of structural and functional characteristics to a widerange of biological constituents, such as the cell membranes. PUFAs areessential for the proper development, maintenance and repair of tissue.Other biological functions of PUFAs include their involvement in theexpression of some genes and their role as precursor molecules forconversion into biologically active metabolites that regulate criticalphysiological functions. Consequently, a lack of, or imbalance in, PUFAlevels has been attributed to certain pathological conditions. FIGS. 1,2 and 3 show the required desaturation and elongation steps for theproduction of long chain fatty acids in the n-3, n-6 and n-9/n-7 PUFAfamilies, respectively. Fatty acid chain elongation systems have beenfound in liver, brain, kidney, lung, adrenals, retina, testis, smallintestine and blood cells, namely leukocytes (Cinti et al., 1992, Prog.Lipid Res., 31: 1-51).

Elongase genes have been identified in Arabidopsis (James et al., 1995,Plant Cell, 7: 309-319 and in C. elegans (WO 00/55330, September, 2000,Napier J. A.). Three separate elongase genes, ELO1, ELO2 and ELO3, havebeen identified from S. cerevisiae. ELO1 elongates myristic acid topalmitic acid (Toke D. A. and Martin C. E., 1996, J. Biol. Chem., 271:18413-18422) while ELO2 and ELO3 elongate long chain saturated fattyacids (Oh et al., 1997, J. Biol. Chem., 272: 17376-17384).

Deficiencies in polyunsaturated fatty acids (PUFAs) have been associatedwith a number of diseases such as eczema, cardiovascular disorders,inflammation, psychiatric disorders, cancer, cystic fibrosis,pre-menstrual syndrome and diabetes (Horrobin D. F. [ed.], 1990, Omega-6Essential Fatty Acids. Pathophysiology and Roles in Clinical Medicine,Wiley-Liss, N.Y. and Mazza G. and Domah B. D. [eds.], 2000, Herbs,Botanicals and Teas, Technomic Publishers, Lancaster, Pa.). Dietssupplemented with PUFAs have been attempted as a treatment for a numberof these conditions. The level of success for such applications hasvaried considerably.

Low levels of linoleic acid (18:2n-6, LA), dihomogamma-linolenic acid(20:3n-6, DCLA) and arachidonic acid (20:4n-6, AA) in adipose tissue ofmales have been correlated with increased mortality from coronary heartdisease (Riemersma et al., 1986, Br. Med. J. [Clin. Res. Ed.], 292:1423-1427). The supplementation of LA and alpha-linolenic acid (18:3n-3,ALA) to patients suffering from hypertension did not increase the tissuelevels of AA or eicosapentaenoic acid (20:5n-3, EPA) which indicatesdefective desaturation and elongation in the n-6 and n-3 fatty acidsystems (Singer et al., 1984, Prostaglandins Leukot. Med., 15: 159-165).Misoprostol, a prostaglandin E1 (PGE1) analogue, has been successfullyused to treat peripheral vascular disease (Goszcz et al., 1998, MethodsFind. Exp. Clin. Pharmacol., 20: 439-445). PGE1 is a cyclooxygenaseproduct of DGLA.

It has been observed that PUFAs can alleviate and correct some of thesymptoms of diabetic neuropathy (Dines et al., 1993, Diabetologia, 36:1132-1138 and Cotter et al., 1995, Diabetic Neuropathy. New Concepts andInsights, Elsevier Science B. V., Amsterdam, pp. 115-120). Researchershave speculated that the production or modulation of the cyclooxygenaseand lipoxygenase metabolites of the n-3 and n-6 fatty acid families isresponsible for some of these beneficial effects.

Most of the lipid metabolism disorders are characterized by a deficiencyin essential fatty acids. This deficiency has been attributed to alteredrate-limiting steps of delta-6-desaturation (D6D) and/ordelta-5-desaturation (D5D) in PUFA biosynthesis.

SUMMARY OF INVENTION

The present invention teaches an isolated polynucleotide sequence,comprising a polynucleotide sequence which is selected from the groupconsisting of: (a) a sequence comprising SEQ ID NO: 4 (ELG4); (b) asequence comprising SEQ ID NO: 8 (ELG6); (c) a sequence comprising SEQID NO: 11 (ELG7); (d) a sequence which is at least 80% homologous with asequence of any of (a) to (c); (e) a sequence which is at least 90%homologous with a sequence of any of (a) to (c); (f) a sequence which isat least 95% homologous with a sequence of any of (a) to (c); (g) asequence which is at least 98% homologous with a sequence of any of (a)to (c); (h) a sequence which is at least 99% homologous with a sequenceof any of (a) to (c); and; (i) a sequence which hybridizes to any of (a)to (h) under stringent conditions. The isolated polynucleotide sequencemay be cDNA. The invention also teaches an isolated polypeptidecomprising an isolated polypeptide selected from the group consistingof: (a) a sequence comprising SEQ ID NO: 5 (ELG4); (b) a sequencecomprising SEQ ID NO: 9 (ELG6); (c) a sequence comprising SEQ ID NO: 12(ELG7); (d) a sequence which is at least 80% homologous with a sequenceof any of (a) to (c); (e) a sequence which is at least 90% homologouswith a sequence of any of (a) to (c); (f) a sequence which is at least95% homologous with a sequence of any of (a) to (c); (g) a sequencewhich is at least 98% homologous with a sequence of any of (a) to (c);and (h) a sequence which is at least 99% homologous with a sequence ofany of (a) to (c).

The invention teaches an isolated polynucleotide sequence, comprising apolynucleotide sequence which is selected from the group consisting of:(a) a sequence comprising SEQ ID NO: 1 (control region for ELG1); (b) asequence comprising SEQ ID NO: 2 (control region for ELG2); (c) asequence comprising SEQ ID NO: 3 (control region for ELG3); (d) asequence comprising SEQ ID NO: 6 (control region for ELG4); (e) asequence comprising SEQ ID NO: 7 (control region for ELG5); (f) asequence comprising SEQ ID NO: 10 (control region for ELG6); (g) asequence comprising SEQ ID NO: 13 (control region for ELG7); (h) asequence which is at least 80% homologous with a sequence of any of (a)to (g); (i) a sequence which is at least 90% homologous with a sequenceof any of (a) to (g); (3) a sequence which is at least 95% homologouswith a sequence of any of (a) to (g); (k) a sequence which is at least98% homologous with a sequence of any of (a) to (g); (l) a sequencewhich is at least 99% homologous with a sequence of any of (a) to (g);and; (m) a sequence which hybridizes to any of (a) to (l) understringent conditions.

The invention includes an isolated polynucleotide fragment selected fromthe group consisting of: (a) a sequence having at least 15 sequentialbases of nucleotides of a sequence of the invention; (b) a sequencehaving at least 30 sequential bases of nucleotides of a sequence of theinvention; and (c) a sequence having at least 50 sequential bases ofnucleotides of a sequence of the invention. The invention includes apolypeptide sequence which retains substantially the same biologicalfunction or activity as or is a functional derivative of a polypeptidesequence of the invention.

The invention includes an isolated polynucleotide sequence, comprising apolynucleotide sequence which retains substantially the same biologicalfunction or activity as or is a functional derivative of apolynucleotide sequence of the invention.

The invention also teaches a vector comprising a polynucleotide sequenceof the invention in a suitable vector. The vector may be heterologous tothe sequence. The vector may contain or encode a tag. The invention alsoteaches a host cell comprising a polynucleotide sequence of theinvention in a host cell which is heterologous to the sequence.

The invention teaches a method for identifying a compound which inhibitsor promotes the activity of a polynucleotide sequence of the invention,comprising the steps of: (a) selecting a control animal having thesequence and a test animal having the sequence; (b) treating the testanimal using a compound; and, (c) determining the relative quantity ofan expression product of the sequence, as between the control animal andthe test animal.

The invention also teaches a method for identifying a compound whichinhibits or promotes the activity of a polynucleotide sequence of theinvention, comprising the steps of: (a) selecting a host cell of theinvention; (b) cloning the host cell and separating the clones into atest group and a control group; (c) treating the test group using acompound; and (d) determining the relative quantity of an expressionproduct of the sequence, as between the test group and the controlgroup.

The invention further teaches a method for identifying a compound whichinhibits or promotes the activity of a polynucleotide sequence of theinvention, comprising the steps of: (a) selecting a test group having ahost cell of the invention or a part thereof, and selecting a suitablecontrol group; (b) treating the test group using a compound; and (c)determining the relative quantity or relative activity of a product ofthe sequence or of the sequence, as between the test group and thecontrol group.

The invention teaches a process for producing a polypeptide sequence ofthe invention comprising the step of culturing the host cell of theinvention under conditions sufficient for the production of thepolypeptide.

The invention teaches a method for identifying a compound which inhibitsor promotes the activity of a polypeptide sequence of the invention,comprising the steps of: (a) selecting a control animal having thesequence and a test animal having the sequence; (b) treating the testanimal using a compound; (c) determining the relative quantity orrelative activity of an expression product of the sequence or of thesequence, as between the control animal and the test animal.

The invention also teaches a method for identifying a compound whichinhibits or promotes the activity of a polypeptide sequence of theinvention, comprising the steps of: (a) selecting a host cell of theinvention; (b) cloning the host cell and separating the clones into atest group and a control group; (c) treating the test group using acompound; and (d) determining the relative quantity or relative activityof an expression product of the sequence or of the sequence, as betweenthe test group and the control group.

The invention includes a method for identifying a compound whichinhibits or promotes the activity of a polypeptide sequence of theinvention, comprising the steps of: (a) selecting a test group having ahost cell of the invention or a part thereof, and selecting a suitablecontrol group; (b) treating the test group using a compound; and (c)determining the relative quantity or relative activity of a product ofthe sequence or of the sequence, as between the test group and thecontrol group.

The invention includes a method for identifying a compound whichmodulates a biological activity of a polypeptide sequence of theinvention, comprising the steps of: (a) providing an assay whichmeasures a biological activity of a polypeptide sequence of theinvention; (b) treating the assay with a compound; and (c) identifying achange in the biological activity of the polypeptide, wherein adifference between the treated assay and a control assay identifies thecompound as modulator of the polypeptide. The polypeptide in this assaymay be provided in a purified, reconstituted, cell extract or whole cellassay format, as required to assay the biological activity in question.

The invention also teaches a method for identifying a compound whichinhibits or promotes the activity of a polynucleotide sequence of theinvention, comprising the steps of: (a) selecting a host cell of theinvention; (b) cloning the host cell and separating the clones into atest group and a control group; (c) treating the test group using acompound; and (d) determining the relative quantity of an expressionproduct of an operably linked polynucleotide to the sequence, as betweenthe test group and the control group.

The invention also teaches a method for identifying a compound whichinhibits or promotes the activity of a polynucleotide sequence of theinvention, comprising the steps of: (a) selecting a test group having ahost cell of the invention or a part thereof, and selecting a suitablecontrol group; (b) treating the test group using a compound; and (c)determining the relative quantity of an expression product of anoperably linked polynucleotide to the sequence, as between the testgroup and the control group.

The invention includes a composition for treating a PUFA disordercomprising a compound which modulates a sequence of the invention and apharmaceutically acceptable carrier. The invention includes the use of acomposition of the invention for treating PUFA disorders.

The invention includes a method for diagnosing the presence of or apredisposition for a PUFA disorder in a subject by detecting a germlinealteration in a sequence of the invention in the subject, comprisingcomparing the germline sequence of a sequence of the invention from atissue sample from the subject with the germline sequence of a wild-typeof the sequence, wherein an alteration in the germline sequence of thesubject indicates the presence of or a predisposition to the PUFAdisorder. The invention teaches a method for diagnosing the presence ofor a predisposition for a PUFA disorder in a subject, comprisingcomparing the sequence of a polypeptide of the invention from a tissuesample from the subject with the sequence of a wild-type of thepolypeptide, wherein an alteration in the sequence of the subject ascompared to the wild-type indicates the presence of or a predispositionto the PUFA disorder.

The invention also teaches a method for identifying a compound whichmodulates a PUFA disorder, comprising identifying a compound whichmodulates the activity of a polynucleotide, wherein the polynucleotideis a coding sequence selected from the group consisting of ELG1, ELG2,ELG3, ELG4, ELG5, ELG6 and ELG7, comprising the steps of: (a) selectinga control animal having the polynucleotide and a test animal having thepolynucleotide; (b) treating the test animal using a compound; and, (c)determining the relative quantity of an expression product of thepolynucleotide, as between the control animal and the test animal.

The invention further teaches a method for identifying a compound whichmodulates a PUFA disorder, comprising identifying a compound whichmodulates the activity of a polynucleotide, wherein the polynucleotideis a coding sequence selected from the group consisting of ELG1, ELG2,ELG3, ELG4, ELG5, ELG6 and ELG7, comprising the steps of: (a) selectinga host cell having the polynucleotide, wherein the host cell isheterologous to the polynucleotide; (b) cloning the host cell andseparating the clones into a test group and a control group; (c)treating the test group using a compound; and (d) determining therelative quantity of an expression product of the polynucleotide, asbetween the test group and the control group.

The invention further teaches a method for identifying a compound whichmodulates a PUFA disorder, comprising identifying a compound whichmodulates the activity of a polynucleotide, wherein the polynucleotideis a coding sequence selected from the group consisting of ELG1, ELG2,ELG3, ELG4, ELG5, ELG6 and ELG7, comprising the steps of:

(a) selecting a test group having a host cell with the polynucleotide ora portion of the host cell, and selecting a suitable control group; (b)treating the test group using a compound; and (c) determining therelative quantity or relative activity of a product of thepolynucleotide or of the poly-nucleotide, as between the test group andthe control group.

The invention teaches a method for identifying a compound modulates aPUFA disorder, comprising identifying a compound which modulates theactivity of a polypeptide selected from the group consisting of ELG1,ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, comprising the steps of: (a)selecting a control animal having the polypeptide and a test animalhaving the polypeptide; (b) treating the test animal using a compound;(c) determining the relative quantity or relative activity of anexpression product of the polypeptide or of the polypeptide, as betweenthe control animal and the test animal.

The invention further teaches a method for identifying a compound whichmodulates a PUFA disorder, comprising identifying a compound whichmodulates the activity of a polypeptide selected from the groupconsisting of ELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, comprisingthe steps of: (a) selecting a host cell comprising the polypeptide,wherein the host cell is heterologous to the polypeptide; (b) cloningthe host cell and separating the clones into a test group and a controlgroup; (c) treating the test group using a compound; and (d) determiningthe relative quantity or relative activity of an expression product ofthe polypeptide or of the polypeptide, as between the test group and thecontrol group.

The invention also teaches a method for identifying a compound whichmodulates a PUFA disorder, comprising identifying a compound whichmodulates the activity of a polypeptide selected from the groupconsisting of ELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, comprisingthe steps of: (a) selecting a test group having a host cell with thepolynucleotide or a portion of the host cell, and selecting a suitablecontrol group; (b) treating the test group using a compound; and (c)determining the relative quantity or relative activity of a product ofthe polypeptide or of the polypeptide, as between the test group and thecontrol group. The invention further teaches a method for identifying acompound which modulates the activity of a polynucleotide, wherein thepolynucleotide is a control region of a gene selected from the groupconsisting of ELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, comprisingthe steps of: (a) selecting a control animal having the polynucleotideand a test animal having the polynucleotide; (b) treating the testanimal using a compound; and, (c) determining the relative quantity ofan expression product of an operably linked polynucleotide to thepolynucleotide, as between the control animal and the test animal. Theanimals of the invention may be mammals. The mammals may be rats.

The invention also teaches a method for identifying a compound whichmodulates the activity of a polynucleotide, wherein the polynucleotideis a control region of a gene selected from the group consisting ofELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, comprising the steps of:(a) selecting a host cell comprising the polynucleotide, wherein thehost cell is heterologous to the polynucleotide; (b) cloning the hostcell and separating the clones into a test group and a control group;(c) treating the test group using a compound; and (d) determining therelative quantity of an expression product of an operably linkedpolynucleotide to the polynucleotide, as between the test group and thecontrol group.

The invention further teaches a method for identifying a compound whichmodulates the activity of a polynucleotide, wherein the polynucleotideis a control region of a gene selected from the group consisting ofELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, comprising the steps of:(a) selecting a test group having a host cell with the polynucleotide ora portion of the host cell, and selecting a suitable control group; (b)treating the test group using a compound; and (c) determining therelative quantity of an expression product of an operably linkedpolynucleotide to the polynucleotide, as between the test group and thecontrol group.

The invention includes a composition for treating a PUFA disordercomprising a compound which modulates a polynucleotide from the codingsequence selected from the group consisting of ELG1, ELG2, ELG3, ELG4,ELG5, ELG6, and ELG7, and a pharmaceutically acceptable carrier.

The invention further teaches a composition for treating a PUFA disordercomprising a compound which modulates a polypeptide selected from thegroup consisting of ELG1, ELG2, ELG3, ELG4, ELG5, ELG6, and ELG7, and apharmaceutically acceptable carrier.

The invention further teaches a composition for treating a PUFA disordercomprising a compound which modulates a polynucleotide from the controlregion selected from the group consisting of ELG1, ELG2, ELG3, ELG4,ELG5, ELG6, and ELG7, and a pharmaceutically acceptable carrier.

The compound may be selected from the group consisting of antibodiesagainst ELG1, ELG2, ELG3 and ELG5.

The invention includes method for diagnosing the presence of or apredisposition for a PUFA disorder in a subject by detecting a germlinealteration in a polynucleotide representing the coding sequence selectedfrom the group consisting of ELG1, ELG2, ELG3, ELG4, ELG5, ELG6, andELG7, from the subject, comprising comparing the germline sequence ofthe polynucleotide from a tissue sample from the subject with thegermline sequence of a wild-type of the polynucleotide, wherein analteration in the germline sequence of the subject indicates thepresence of or a predisposition to the PUFA disorder.

The invention also teaches method for diagnosing the presence of or apredisposition for a PUFA disorder in a subject by detecting a germlinealteration in a polynucleotide representing the control region selectedfrom the group consisting of ELG1, ELG2, ELG3 and ELG5 in the subject,comprising comparing the germline sequence of the polynucleotide from atissue sample from the subject with the gerrnline sequence of awild-type of the polynucleotide, wherein an alteration in the germlinesequence of the subject indicates the presence of or a predisposition tothe PUFA disorder.

The invention also teaches a method for diagnosing the presence of or apredisposition for a PUFA disorder in a subject, comprising comparingthe sequence of a polypeptide selected from the group consisting ofELG1, ELG2, ELG3, ELG4, ELG5, ELG6, and ELG7, from the subject with thesequence of a wild-type of the polypeptide, wherein an alteration in thesequence of the subject as compared to the wild-type indicates thepresence of or a predisposition to the PUFA disorder.

The invention further teaches a method for identifying a compound whichinhibits or promotes the overall activity of two or morepolynucleotides, wherein the polynucleotides are control regions of twoor more different genes selected from the group consisting of ELG1,ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, comprising the steps of: (a)selecting a host cell having the polynucleotides, wherein the host cellis heterologous to the polynucleotides; (b) cloning the host cell andseparating the clones into a test group and a control group; (c)treating the test group using a compound; and (d) determining therelative quantities of expression products of operably linkedpolynucleotides to the polynucleotides, as between the test group andthe control group.

The invention further teaches a method for identifying a compound whichinhibits or promotes the overall activity of two or morepolynucleotides, wherein the polynucleotides are from control regions ofthe polynucleotides, selected from the group consisting of ELG1, ELG2,ELG3, ELG4, ELG5, ELG6 and ELG7, comprising the steps of: (a) selectinga test group having a host cell with the polynucleotide or a portion ofthe host cell, and selecting a suitable control group; (b) treating thetest group using a compound; and (c) determining the relative quantitiesof expression products of operably linked polynucleotides to thepolynucleotides, as between the test group and the control group.

The invention teaches a method for identifying a compound which inhibitsor promotes the activity of two or more polynucleotides, wherein thepolynucleotides are coding sequences selected from the group consistingof ELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, operably associated withpromoter regions, wherein the promoter regions are effective toinitiate, terminate or regulate the level of expression of the nucleicacid sequence, comprising the steps of: (a) selecting a host cell havingthe polynucleotides, wherein the host cell are heterologous to thepolynucleotides; (b) cloning the host cell and separating the clonesinto a test group and a control group; (c) treating the test group usinga compound; and (d) determining the relative quantity or relativeactivity of an expression product of the polynucleotide, as between thetest group and the control group.

The invention further teaches a method for identifying a compound whichinhibits or promotes the activity of two or more polynucleotides,wherein the polynucleotides are coding sequences selected from the groupconsisting of ELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, operablyassociated with promoter regions, wherein the promoter regions areeffective to initiate, terminate or regulate the level of expression ofthe nucleic acid sequence, comprising the steps of: (a) selecting a testgroup having a host cell with the polynucleotide or a portion of thehost cell, and selecting a suitable control group; (b) treating the testgroup using a compound; and (c) determining the relative quantity orrelative activity of an expression product of the polynucleotide, asbetween the test group and the control group.

The invention includes a method for identifying a compound whichinhibits or promotes the activity of a mammalian delta-5-desaturaseenzyme and one or more enzymes selected from the group consisting ofELG1, ELG2, ELG3, ELG4, ELG5, ELG6 or ELG7, within the same host system,comprising the steps of: (a) providing a host system containing nucleicacid sequences which encode for a mammalian delta-5-desaturase and oneor more mammalian elongase enzymes selected from the group consisting ofELG1, ELG2, ELG3, ELG4, ELG5, ELG6 or ELG7, operably associated withpromoter regions, wherein the promoter regions are effective toinitiate, terminate or regulate the level of expression of the nucleicacid sequence; (b) contacting the host system with a test component; (c)simultaneously evaluating the enzymatic activities of thedelta-5-desaturase and the elongase enzymes, wherein a measurabledifference in a level of lipid metabolites or associated cofactors inthe presence of the test component compared to a control under identicalconditions but in the absence of the test component is an indicator ofthe ability of the test component to modulate delta-5-desaturase and/orelongase enzyme activity; and (d) identifying as the compound a testcomponent which exhibits the ability.

The invention further teaches a method for identifying a compound whichinhibits or promotes the activity of a mammalian delta-6-desaturaseenzyme and one or more enzymes selected from the group consisting ofELG1, ELG2, ELG3, ELG4, ELG5, ELG6 or ELG7, within the same host system,comprising the steps of: (a) providing a host system containing nucleicacid sequences which encode for a mammalian delta-6-desaturase and oneor more mammalian elongase enzymes selected from the group consisting ofELG1, ELG2, ELG3, ELG4, ELG5, ELG6 or ELG7, operably associated withpromoter regions, wherein the promoter regions are effective toinitiate, terminate or regulate the level of expression of the nucleicacid sequence; (b) contacting the host system with a test component; (c)simultaneously evaluating the enzymatic activities of thedelta-6-desaturase and the elongase enzymes, wherein a measurabledifference in a level of lipid metabolites or associated cofactors inthe presence of the test component compared to a control under identicalconditions but in the absence of the test component is an indicator ofthe ability of the test component to modulate delta-6-desaturase and/orelongase enzyme activity; and (d) identifying as the compound a testcomponent which exhibits the ability.

The invention teaches a method for identifying a compound which inhibitsor promotes the activity of a mammalian delta-5- and delta-6-desaturaseenzyme and/or one or more enzymes selected from the group consisting ofELG1, ELG2, ELG3, ELG4, ELG5, ELG6 or ELG7, within the same host system,comprising the steps of: (a) providing a host system containing nucleicacid sequences which encode simultaneously for a mammaliandelta-5-clesaturasc, a mammalian delta-6-desaturase and one or moremammalian elongase enzymes selected from the group consisting of ELG1,ELG2, ELG3, ELG4, ELG5, ELG6 or ELG7, operably associated with promoterregions, wherein the promoter regions are effective to initiate,terminate or regulate the level of expression of the nucleic acidsequence; (b) contacting the host system with a test component; (c)simultaneously evaluating the enzymatic activities of thedelta-5-desaturase, the delta-6-desaturase and the elongase enzymes,wherein a measurable difference in a level of lipid metabolites orassociated cofactors in the presence of the test component compared to acontrol under identical conditions but in the absence of the testcomponent is an indicator of the ability of the test component tomodulate delta-5- and/or delta-6-desaturase and/or elongase enzymeactivity; and (d) identifying as the compound a test component whichexhibits the ability.

The invention includes a composition for treating a PUFA disordercomprising a compound which modulates two or more human polynucleotidesfrom control regions selected from the group consisting of ELG1, ELG2,ELG3, ELG4, ELG5, ELG6, ELG7, delta-5-desaturase, delta-6-desaturase anda pharmaceutically acceptable carrier.

The invention includes a method for detecting the presence of or thepredisposition for a PUFA disorder, the method comprising determiningthe level of expression of two or more expression products of genesselected from the group consisting of ELG1, ELG2, ELG3, ELG4, ELG5,ELG6, ELG7, delta-5-desaturase, delta-6-desaturase, in a subjectrelative to a predetermined control level of expression, wherein anymodified expression of the expression products as compared to thecontrol is indicative of the presence of or the predisposition for aPUFA disorder.

The invention further includes an antibody immunoreactive with apolypeptide of the invention or an immunogenic portion thereof. Theinvention includes an antibody immunoreactive with an elongasepolypeptide selected from the group consisting of ELG1. ELG2, ELG3,ELG4, ELG5, ELG6 and ELG7, or an immunogenic portion thereof.

The invention teaches a method for screening a medium for an elongasepolypeptide of the invention or selected from the group consisting ofELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, comprising: (a) labellingan antibody of the invention with a marker molecule to form a conjugate;(b) exposing the conjugate to the medium; and (c) determining whetherthere is binding between the conjugate and a biomolecule in the medium,wherein the binding indicates the presence of the polypeptide.

The invention teaches a method for screening a medium for an elongasepolypeptide of the invention or selected from the group consisting ofELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7, comprising; (a) exposing anantibody of the invention to the medium; (b) exposing the antibody to amarker molecule; and (c) determining whether there is binding betweenthe marker molecule and a biomolecule in the medium, wherein the bindingindicates the presence of the polypeptide.

The invention includes compounds identified by the method of theinventions.

The invention further includes a method for diagnosing the presence ofor a predisposition for a PUFA disorder in a subject by detectingalterations in the elongation of PUFA in a peripheral blood leukocyteobtained from the subject. The invention includes a method formonitoring the development of a PUFA disorder in a subject by detectingalterations in the elongation of PUFA in a peripheral blood leukocyteobtained from the subjects. The invention further teaches a method forassessing the effect of test compounds on a PUFA disorder in a subjectby assessing alterations in the elongation of PUFA in a peripheral bloodleukocyte obtained from the subject.

The compounds of the invention may be selected from the group consistingof small organic molecules, peptides, polypeptides, antisense molecules,oligonucleotides, polynucleotides, fatty acids and derivatives thereof.

The invention further teaches the use of pebulate sulphoxide for thetreatment of a disorder of the invention.

The disorders of the invention may be selected from the group consistingof peripheral cardiovascular disease, coronary heart disease,hypertension, atopic eczema, rheumatoid arthritis, Sjögren's syndrome,gastrointestinal disorders, viral diseases and postviral fatigue,psychiatric disorders, pre-menstrual syndrome, endometriosis, cysticfibrosis, alcoholism, congenital liver disease, Alzheimer's syndrome,cancer, diabetes and diabetic complications. The disorders of theinvention may be selected from the group consisting of eczema,cardiovascular disorders (including but not limited toDhypertriglyceridemia, dyslipidemia, atherosclerosis, coronary arterydisease, cerebrovascular disease hypertension, and peripheral vasculardisease), inflammation (including but not limited to sinusitis, asthma,pancreatitis, osteoarthritis, rheumatoid arthritis and acne), Sjögren'ssyndrome, gastrointestinal disorders, viral diseases and postviralfatigue, body weight disorders (including but not limited to obesity,cachexia and anorexia), psychiatric disorders, cancer, cystic fibrosis,endometriosis, pre-menstrual syndrome, alcoholism, congenital liverdisease, Alzheimer's syndrome, hypercholesterolemia, autoimmunedisorders, atopic disorders, acute respiratory distress syndrome,articular cartilage degradation, diabetes and diabetic complications.

BRIEF DESCRIPTION OF THE DRAWINGS

In the following description, the invention will be explained in detailwith the aid of the accompanying figures, which illustrate preferredembodiments of the present invention and in which:

FIG. 1 is a schematic diagram of the n-3 fatty acid metabolic pathways;

FIG. 2 is a schematic diagram of the n-6 fatty acid metabolic pathways;

FIG. 3 is a schematic diagram of the n-9 and n-7 fatty acid metabolicpathways;

FIG. 4 is a chart showing a multiple alignment among the 7 humanelongases (ELG1 (SEQ. ID. NO. 59), ELG2 (SEQ. ID. NO. 60), ELG3 (SEQ.ID. NO. 61), ELG4 (SEQ. ID. NO. 5), ELG5 (SEQ. ID. NO. 62), ELG6 (SEQ.ID. NO. 9), and ELG7 (SEQ. ID. NO. 12)), highlighting the invariantresidues (marked by asterisks), the histidine box (marked by a box) andthe ER retention signals (marked by boxes);

FIG. 5 is a graph illustrating the Transmembrane Hidden Markov Model(TMHMM) prediction for transmembrane regions for ELG7;

FIG. 6 is a diagram showing a topological model of a human elongaseembedded in the endoplasmic reticulum;

FIG. 7 is a schematic representation of plasmid pTh1009.1 (6744 bp). Thehuman elongase (ELG1) coding sequence is shown between restriction sitesfor KpnI and NotI;

FIG. 8 shows the nucleotide sequence of the control region of ELG1between position −1877 and −2865 from the translation initiation codon,ATG. This figure corresponds to SEQ. ID. NO. 1;

FIG. 9 shows the nucleotide sequence of the control region of ELG2between position −53118 and −53626 from the translation initiationcodon, ATG. This figure corresponds to SEQ. ID. NO. 2;

FIG. 10 shows the nucleotide sequence of the control region of ELG3between position −37 and −1381 from the translation initiation codon,ATG. This figure corresponds to SEQ. ID. NO. 3;

FIG. 11 shows the nucleotide sequence and amino acid sequence of theELG4 gene. This figure corresponds to SEQ. ID. NOS. 4 and 5;

FIG. 12 shows a 2456 bp fragment of the nucleotide sequence of thecontrol region of ELG4. This figure corresponds to SEQ. ID. NO. 6;

FIG. 13 shows the nucleotide sequence of the control region of ELG5between position −1 and −1411 from the translation initiation codon,ATG. This figure corresponds to SEQ. ID. NO. 7;

FIG. 14 shows the nucleotide sequence and amino acid sequence of theELG6 gene. This figure corresponds to SEQ. ID. NOS. 8 and 9;

FIG. 15 shows the nucleotide sequence of the control region of ELG6between position −1 and −1937 from the translation initiation codon,ATG. This figure corresponds to SEQ. ID. NO. 10;

FIG. 16 shows the nucleotide sequence and amino acid sequence of theELG7 gene. This figure corresponds to SEQ. ID. NOS. 11 and 12;

FIG. 17 shows the nucleotide sequence of the control region of ELG7between position −1 and −2000 from the translation initiation codon,ATG. This figure corresponds to SEQ. ID. NO. 13;

FIG. 18 is a schematic representation of plasmid pTh1109.2 (6743 bp).The human elongase (ELG1) coding sequence is shown between restrictionsites for KpnI and NotI;

FIG. 19 is a schematic representation of plasmid pLh5015.1 (7927 bp).The human elongase (ELG3) coding sequence is shown between restrictionsites for BamHI and XbaI;

FIG. 20 is a schematic representation of plasmid pGh3020.1 (6168 bp).The control region for human elongase (ELG3) is shown between two BglIIrestriction sites:

FIG. 21 shows an HPLC analysis of radiolabelled methyl esters of fattyacids from yeast transformed with pTh1021.1 incubated with[1-¹⁴C]18:3n-6, [1-¹⁴C]20:4n-6, [1-¹⁴C]18:3n-3 and [1-¹⁴C]20:5n-3;

FIG. 22 shows an HPLC analysis of radiolabelled methyl esters of fattyacids from yeast transformed with pYES2/CT incubated with[1-¹⁴C]18:3n-6, [1-¹⁴C]20:4n-6. [1-¹⁴C]18:3n-3 and [1-¹⁴C]20:5n-3;

FIG. 23 shows an HPLC analysis of radiolabelled methyl esters of fattyacids from yeast co-expressing D6D/V5-His and ELG3, incubated with[1-¹⁴C]18:2n-6 or [1-¹⁴C]18:3n-3 and with or without galactose;

FIG. 24 shows an HPLC analysis of radiolabelled methyl esters of fattyacids from yeast co-expressing D6D/V5-His and ELG3, incubated with[1-¹⁴C]20:4n-6 or [1-¹⁴C]20:5n-3 and with or without galactose;

FIG. 25 shows an HPLC analysis of radiolabelled methyl esters of fattyacids from yeast co-expressing D5D/V5-His and ELG3, incubated with[1-¹⁴C]20:4n-3 or [1-¹⁴C]20:3n-6 and with or without galactose;

FIG. 26 shows an HPLC analysis of ratholabelled methyl esters of fattyacids from yeast co-expressing D5D/V5-His and ELG3, incubated with[1-¹⁴C]18:2n-6 or [1-¹⁴C]18:3n-3 and with or without galactose.

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, research has indicated that increased levels of LAor DGLA are the result of decreased activities of delta-6 anddelta-5-desaturase enzymes. The present inventors have found evidencethat both the desaturase and elongase activities are affected in a PUFArelated disorder.

The desaturase and elongase enzyme activities in liver microsomes fromstreptozotocin (STZ)-induced diabetic rats was assayed at 2 and 7 weekspost-induction. Table 1 indicates the decrease in activities compared toa control, observed during the course of the experiment. An equivalentdecrease in elongation activity in STZ-induced diabetic rats has beenpreviously reported (Suneja et al., 1990, Biochim. Biophys. Acta, 1042:81-85).

TABLE 1 Percent Decrease of the Desaturase and Elongase Activities inLiver Microsomes from STZ-Induced Diabetic Rats % Decrease ENZYME 2weeks 7 weeks D6D 28 33 (18:2n-6 → 18:3n-6) Elongase 46 43 (18:3n-6 →20:3n-6) D5D 33 41 (20:3n-6 → 20:4n-6)

This data, when considered in view of what is known regarding therelationship between PUFAs and disease (above), indicates that elongasegenes are involved in the development and regulation of lipid associateddiseases such as inflammation, hypercholesterolemia, autoimmunedisorders, atopic disorders, cystic fibrosis, psychiatric disorders,cancer, acute respiratory distress syndrome, articular cartilagedegradation, arthritis, diabetes and diabetic complications. Since PUFAsare involved in a number of cell regulatory processes, the elongasegenes and gene products represent realistic drug targets for thetreatment or prevention of fatty acid associated diseases.

The present inventors used bioinformatic techniques to identify andanalyze 7 human elongase genes (ELG1, ELG2, ELG3, ELG4, ELG5, ELG6 andELG7). The amino acid sequences of the 7 human elongases were comparedusing a ClustalW algorithm (Thompson et al., 1994, Nucl. Acids Res., 22:4673-4680). One highly conserved motif, a histidine box containing 3histidine residues, found also in a number of membrane-bounddesaturases, is common to all 7 sequences. Twenty five other invariantresidues, suggesting their critical importance in the catalytic activityand structure of the elongases, are identified in the multiple alignmentwhere they are indicated by asterisk (see FIG. 4).

Table 2 shows the percent identity among the 7 human elongases. Thepercent identities range from a low of 17% (ELG3/ELG5 and ELG3/ELG6) toa high of 55% (ELG1/ELG4).

TABLE 2 Percent Identities Among the 7 Human Elongases ELG1 ELG2 ELG3ELG4 ELG5 ELG6 ELG7 ELG1 100 ELG2 30 100 ELG3 29 54 100 ELG4 55 31 34100 ELG5 18 18 17 22 100 ELG6 21 18 17 22 43 100 ELG7 33 37 37 36 18 19100

Based on a hidden Markov model for predicting transmembrane regions(Sonnhammer et al., 1998, In Proc. of Sixth Int. Conf. on IntelligentSystems for Molecular Biology, AAAI Press, CA, pp. 175-182), this familyof seven elongases has 7 membrane spanning regions (FIG. 5). Theseregions are highly conserved with respect to position in the amino acidsequences of the 7 elongases. The invariant histidine box is predictedto be embedded in the fourth transmembrane region. This differs fromthat of the membrane-bound desaturases wherein the three conservedhistidine boxes are predicted to be in cytosolic loops (Shanklin et al.,1994, Biochemistry, 33: (12787-12794). The present model for the humanelongases encompasses a ring of transmembrane domains enclosing an innercatalytic cavity for insertion of fatty acyl chains. A proposedtopological model of the elongases embedded in the endoplasmic reticulum(ER) is shown in FIG. 6.

The present inventors have discovered that each of the proteins has anER retention signal (Jackson et al., 1990, EMBO J, 9: 3153-3162 andNilsson T. and Warren G., 1994, Curr. Opin. Cell Biol., 6: 517-521) atthe carboxyl terminus. In ER resident proteins with a type I topology(amino terminus in the lumen), the signal has been shown to consist oftwo critical lysines, which are in a −3 and a −4/−5 position relative tothe carboxyl terminus in their cytosolic, exposed tails (K[X]KXX, whereX is any amino acid). Each of the elongases has such a retention signal.Both ELG2 and ELG5, however, have modified forms of this signal. Usingbioinformatic techniques, the control region of the ELG1 gene wasidentified and mapped out. By searching GenBank's EST division usingBLASTN with genomic DNA and CDS for the ELG1 gene, a number of differentESTs were identified containing 5′ UTR for the gene. There were 2families of such ESTs each arising from different upstream exons whichexclusively contain 5′ UTR. The first exon has its 3′ position at −2306while the second exon has its 3′ position at −1877 from the translationinitiation codon, ATG. A 128 bp fragment of another EST (GenBankAccession No. A1373530) was also identified approximately 2.9 kbupstream of the ATG. The control region between positions −1877 and−2865 from the translation initiation codon, ATG is shown in FIG. 8. Arepetitive element is farther identified upstream of −3600.

Northern blot studies evaluating tissue distribution showed that the˜1.3 kb ELG1 transcript is expressed in all tissues examined, withhighest levels in kidney, brain, heart and placenta.

ELG2 Gene and Polypeptide

BLASTP of the GenBank NR database with yeast ELO1 identified a proteinwith unknown function (GenBank Accession No. CAB41293, since withdrawn)as a potential elongase. This protein sequence was deduced from genomicDNA (GenBank Accession No. AL034374) and represents only a partialsequence. Using GeneTrapper technology (Gibco BRL) the complete codingsequence of this protein, termed ELG2 by the present inventors, wascloned and the nucleotide sequence determined by DNA sequencing. Sincethen, the ELG2 coding sequence and deduced protein sequence have beensubmitted to GenBank (Accession Nos. AF231981 and AAF70631,respectively).

The cDNA coding for ELG2 was obtained by PCR and cloned into the yeastexpression vector pYES2/CT. The sequence was verified by DNA sequencingand the resulting plasmid was designated pTh1014.1.

Yeast cells transformed with pTh1014.1 and expressing ELG2 were shown toelongate 18:3n-6 to 20:3n-6 and 22:3n-6, 20:4n-6 to 22:4n-6 and 24:4n-6,18:3n-3 to 20:3n-3, and 20:5n-3 to 22:5n-3 (refer to Table 3 in Example19). Yeast transformed with the pYES2/CT vector did not elongate any ofthese substrates. This proved that the ELG2 gene encodes a PUFAelongase. It has been reported that this gene, referred to as HELO orHSELO, encodes a protein that is involved in the elongation of a varietyof PUFAs including 18:3n-6, 20:4n-6, 18:4n-3, 20:5n-3 and 18:3n-3(Leonard et al., 2000, Biochem. J, 350: 765-770 and Mukerji et al., PCTApplication WO 00/12720).

Exons for ELG2 were mapped onto genomic DNA from human chromosome 6(GenBank Accession No. AL034374). The gene was found to comprise 7coding exons spanning 26.5 kb.

Using bioinformatic techniques, the control region of the ELG2 gene wasidentified and mapped out. Using sequence data from the presentinventors' clones obtained by GeneTrapper technology, 5′ UTR wasidentified in an exon approximately 53 kb upstream of the ATG. Thisfinding was corroborated by searching GenBank's EST division usingBLASTN with the ELG2 CDS. Two ESTs were identified (GenBank AccessionNos. AA282396 and BE779576) which mapped to the same upstream exon. Thecontrol region between positions −53118 and −53626 from the translationinitiation codon, ATG is shown in FIG. 9. Sequence from which an EST isderived (GenBank Accession No. AA557341) lies immediately upstream ofthis region. A repetitive element is identified approximately 1.4 kbfurther upstream from the 3′ end of this 5′ UTR-containing exon.

Northern blot studies evaluating tissue distribution showed that the 2.8kb ELG2 transcript is expressed in all tissues examined, with highestlevels in brain, heart and kidney, and moderate levels in the liver.

ELG3 Gene and Polypeptide

BLASTP of the GenBank NR database with yeast ELO1 identified a proteinwith unknown function (GenBank Accession No. BAA91096), as a potentialelongase. This protein was deduced from cDNA (GenBank Accession No.AK000341) and is termed ELG3 by the present inventors.

The cDNA coding for ELG3 was obtained by PCR and cloned into the yeastexpression vector pYES2/CT. The nucleotide sequence was verified by DNAsequencing and the resulting plasmid was designated pTh1015.1. Incomparison to GenBank Accession No. BAA91096, the protein encoded by theELG3 gene contains two amino acid substitutions, T31M and V1791.

Yeast cells transformed with pTh015.1 and expressing ELG3 were shown toelongate 18:3n-6 to 20:3n-6, 20:4n-6 to 22:4n-6 and 24:4n-6, 18:3n-3 to20:3n-3, and 20:5n-3 to 22:5n-3 and 24:5n-3 (refer to Table 3 in Example19). Yeast transformed with the pYES2/CT vector did not elongate any ofthese substrates. This proved that ELG3 encodes a PUFA elongase. Thereis no published data demonstrating that this protein is a PUFA elongase.However, Mukerji et al. (PCT Application WO 00/12720) indicate that anEST (GenBank Accession No. A1815960), found by the present inventors torepresent a portion of the CDS of ELG3, may encode a partial PUFAelongase. They did not clone the coding sequence derived from this ESTnor determine its function.

The mouse ortholog of human ELG3, Ssc2 (GenBank Accession No. AF170908),has been identified as putatively involved in fatty acid elongation.However, enzymatic function has not been confirmed (Tvrdik et al., 2000,J. Cell Biol., 149: 707-717). Mouse Ssc2 is 88% identical and 94%similar to human ELG3.

Exons for ELG3 were mapped onto genomic DNA from human chromosome 6(GenBank Accession No. AL121955). The gene was found to comprise 8coding exons spanning 60.5 kb.

Using bioinformatic techniques, the control region of the ELG3 gene wasidentified and mapped out. By searching GenBank using BLASTN withgenomic DNA and CDS for the ELG3 gene, 2 sequences (GenBank AccessionNos. BE778035 and AK000341) were identified containing 84 bP of 5′ UTRimmediately upstream of the initiation codon, ATG. The control regionbetween positions −37 and −1381 from the translation initiation codon,ATG was cloned (see Example 11) and is shown in FIG. 10.

Northern blot studies evaluating tissue distribution showed that the˜4.4 kb ELG3 transcript is moderately expressed in brain, with lowerlevels in heart, liver and placenta. This transcript was not detected inany of the other tissues examined.

ELG4 Gene and Polypeptide

BLASTP of the GenBank NR database with yeast ELO1 identified a proteinwith unknown function (GenBank Accession No. CAB70777) as a potentialelongase. This protein sequence was deduced from cDNA (GenBank AccessionNo. AL137506) and represents only a partial sequence. Using GeneTrappertechnology (Gibco BRL) and PCR amplification the full coding sequencefor this protein, termed ELG4 by the present inventors, was cloned. ThecDNA sequence was determined by DNA sequencing. The coding sequence andamino sequence of ELG4 are shown in FIG. 11. Since then, Kawakami andcoworkers have submitted a cDNA sequence to GenBank (Accession No.AK027216) that is similar to ELG4. However, in comparison to ELG4 itdoes not contain the first 31 nucleotides of the coding sequence, hasseveral nucleotide substitutions and has a one nucleotide insertion.

The cDNA coding for ELG4 was obtained by PCR and cloned into the yeastexpression vector pYES2/CT. The sequence was verified by DNA sequencingand the resulting plasmid was designated pTh1021.1.

Yeast cells transformed with pTh1021.1 and expressing ELG4 were shown toelongate 18:3n-6 to 20:3n-6 and 22:3n-6, 20:4n-6 to 22:4n-6 and 24:4n-6,and 18:3n-3 to 20:3n-3 and 22:3n-3, and 20:5n-3 to 22:5n-3 and 24:5n-3(Refer to Table 3 in Example 19 and FIG. 21). Yeast transformed with thepYES2/CT vector did not elongate any of these substrates. This provedthat the ELG4 gene encodes a PUFA elongase.

Exons for ELG4 were mapped onto genomic DNA from human chromosome 5(GenBank Accession No. AC021601). The gene was found to comprise 7coding exons spanning at least 32 kb.

Using bioinformatic techniques, the control region of the ELG4 gene wasidentified and mapped out. Using sequence data from the presentinventors' clones obtained by GeneTrapper technology, the 5′ UTR wasidentified in 3 consecutive, alternatively spliced, upstream exons fromthe exon containing the initiation codon, ATG. The most immediateupstream exon is approximately 12 kb upstream, the next exon is over 13kb upstream and the farthest upstream exon is at least 19 kb upstreamfrom the ATG. The control region containing a 2456 bp fragment with itsend at the 3′ end of this first (most upstream) exon is shown in FIG.12. It is flanked at its 5′ end by a repetitive element.

Northern blot studies evaluating tissue distribution showed that the˜4.3 kb ELG4 transcript is highly expressed in kidney and moderatelyexpressed in brain and heart. Low levels of transcript were detected inskeletal muscle, colon, thymus, liver, small intestine and placenta. Thetranscript was not detected in spleen and peripheral blood leukocytes.

ELG5 Gene and Polypeptide

The cDNA sequence of a GenBank entry (Accession No. AK027031) encodesanother potential elongase. The deduced protein sequence (GenBankAccession No. BAB15632) is termed ELG5 by the present inventors.

The cDNA coding for ELG5 was obtained by PCR and cloned into the yeastexpression vector pYES2/CT. The nucleotide sequence was verified by DNAsequencing and the resulting plasmid was designated pTh1018.1.

Yeast cells transformed with pTh1018.1 and expressing ELG5 were shown toconvert 18:3n-6 to 20:3n-6 and 18:3n-3 to 20:3n-3 (refer to Table 3 inExample 19). Yeast cells transformed with the pYES2/CT vector did notelongate either of these substrates. This proved that the ELG5 geneencodes a PUFA elongase. There is no published data demonstrating thatthis protein is a PUFA elongase. Mukerji et al. (PCT Application WO00/12720) indicate that HS3, which is identical to ELG5, might be a PUFAelongase. The coding sequence was cloned, however, enzymatic functionwas not evaluated.

Exons for ELG5 were mapped onto genomic DNA from human chromosome 4(GenBank Accession Nos. AC004050. AC022952 and AP002080). The gene wasfound to comprise 4 coding exons spanning at least 88 kb.

Using bioinformatic techniques, the control region of the ELG5 gene wasidentified and mapped out. By searching GenBank's EST division usingBLASTN with genomic DNA and CDS for the ELG5 gene, a number of differentESTs were identified containing 5′ UTR for the gene. The control regionbetween positions −1 and −1411 from the ATG is shown in FIG. 13. Thisregion is flanked at its 5′ end by a repetitive element.

Northern blot studies evaluating tissue distribution showed twotranscripts for ELG5. The ˜3.0 kb transcript is highly expressed inliver, with moderate expression in brain, colon and kidney, and lowexpression in heart, thymus, small intestine, placenta and skeletalmuscle. The ˜7.6 kb transcript is expressed in moderate levels in thebrain and low levels in colon, kidney and liver.

ELG6 Gene and Polypeptide

ELG6 was identified by searching Homo sapiens sequences in GenBank'sHTGS division with the coding sequences for ELG1, ELG2, ELG3, ELG4 andELG5 using the TBLASTN algorithm. One sequence was identified ascontaining sequences similar to human elongases (GenBank Accession No.AL160011). This approach, however, failed to identify the beginning ofthe gene containing the translation initiation site. Therefore, furthermapping and identification of ELG6 coding sequences was obtained usingCig30 (cold inducible membrane glycoprotein 30) from Mus musculus(GenBank Accession No. U97107), a protein found to be similar to ELG6,as a template. The first coding exon of ELG6 containing the initiationcodon, ATG, was identified in this manner.

The cDNA coding for ELG6 was obtained by PCR and cloned into the yeastexpression vector pYES/CT. The nucleotide sequence was verified by DNAsequencing and the resulting plasmid was designated pTh1041.1. Thecoding sequence and amino sequence of ELG6 are shown in FIG. 14.

Yeast cells transformed with pTh1041.1 and expressing ELG6 were shown toelongate 18:3n-6 to 20:3n-6 and 18:3n-3 to 20:3n-3 (refer to Table 3 inExample 19). Yeast cells transformed with the pYES2/CT vector did notelongate either of these substrates. This proved that the ELG6 geneencodes a PUFA elongase.

The mouse ortholog of human ELG6, Cig30 (GenBank Accession No. U97107),has been implicated in fatty acid elongation due to its ability tocomplement yeast ELO2 mutants. Furthermore, Cig30 gene expressioncorrelates with elongase activity during brown fat recruitment in mice(Tvrdik et al., 1997, J. Biol. Chem., 272: 31738-31746 and Tvrdik etal., 2000, J. Cell Biol., 149: 707-717). Mouse Cig30 is 69% identicaland 81% similar to human ELG6.

Since the inventors' discovery of ELG6 another record has been submittedto GenBank (GenBank Accession No. AF292387) containing genomic DNA and apartial CDS for the Homo sapiens Cig30 ortholog. Sequence annotations,however, do not indicate the presence of the first coding exon.

Exons for ELG6 were mapped onto genomic DNA from human chromosome 10(GenBank Accession No. AL160011). The gene was found to comprise 4coding exons spanning approximately 2.7 kb.

Using bioinformatic techniques, the control region of the ELG6 gene wasidentified and mapped out. The control region between positions −1 and−1937 from the ATG is shown in FIG. 15.

The transcript for ELG6 was not detected in standard Northern blotanalysis in any of the tissues examined.

ELG7 Gene and Polypeptide

ELG7 was identified by searching Homo sapiens sequences in GenBank'sHTGS division with the coding sequences for ELG1, ELG2, ELG3, ELG4 andELG5 using the TBLASTN algorithm. A number of sequences were identifiedcontaining exons with sequences similar to human elongases. One suchsequence, 164 kb in length, (GenBank Accession No. AL132875) was foundby the present inventors to contain a previously unidentified gene,termed ELG7, in 6 coding exons spanning approximately 30.5 kb of genomicDNA. The cDNA coding for ELG7 was obtained by PCR and cloned into theyeast expression vector pYES2/CT. The nucleotide sequence was verifiedby DNA sequencing and the resulting plasmid was designated pTh1044.1.The coding sequence and amino sequence of ELG7 are shown in FIG. 16.

Yeast cells transformed with pTh1044.1 and expressing ELG7 were shown toconvert 18:3n-3 to 20:3n-3 (refer to Table 3 in Example 19). Yeasttransformed with the pYES2/CT vector did not elongate this substrate.This proved that ELG7 encodes a PUFA elongase.

Using bioinformatic techniques, the control region of the ELG7 gene wasidentified and mapped out. By searching GenBank's EST division usingBLASTN with genomic DNA for the ELG7 gene, a human EST containing 118 bpof 5′ UTR for the gene was identified immediately upstream of theinitiation codon, ATG (GenBank Accession No. BE878648). The controlregion between positions −1 and −2000 from the ATG is shown in FIG. 17.A repetitive element is further identified upstream of −2700.

Northern blot studies evaluating tissue distribution showed that the˜3.0 kb ELG7 transcript is expressed in brain, thymus and placenta. Thistranscript was not detected in any of the other tissues examined

Subject Polynucleotides and Polypeptides

The subject polynucleotides and polypeptides may be employed as researchreagents and materials for discovery of treatments of and diagnosticsfor disease, particularly human disease, as further discussed herein.

Nucleotide Probes

The nucleic acid molecules of the invention allow those skilled in theart to construct nucleotide probes for use in the detection ofnucleotide sequences in biological materials. As described herein, anumber of unique restriction sequences for restriction enzymes areincorporated in the nucleic acid molecule identified in the sequencelistings of the subject polynucleotides, and these provide access tonucleotide sequences which code for polypeptides unique to the subjectpolynucleotides of the invention. Nucleotide sequences unique to thesubject polynucleotides or isoforms thereof can also be constructed bychemical synthesis and enzymatic ligation reactions carried out byprocedures known in the art. A nucleotide probe may be labeled with adetectable marker such as a radioactive label which provides for anadequate signal and has sufficient half-life such as ³²P, ³H, ¹⁴C or thelike. Other detectable markers which may be used include antigens thatare recognized by a specific labeled antibody, fluorescent compounds,enzymes, antibodies specific for a labeled antigen, and chemiluminescentcompounds. An appropriate label may be selected with regard to the rateof hybridization and binding of the probe to the nucleotide to bedetected and the amount of nucleotide available for hybridization. Thenucleotide probes may be used to detect genes related to or analogous tothe subject polynucleotides of the invention. Accordingly, the presentinvention also provides a method of detecting the presence of nucleicacid molecules encoding a polypeptide related to or analogous to thesubject polynucleotides in a sample comprising contacting the sampleunder hybridization conditions with one or more of the nucleotide probesof the invention labeled with a detectable marker, and determining thedegree of hybridization between the nucleic acid molecule in the sampleand the nucleotide probes.

Hybridization conditions which may be used in the method of theinvention are known in the art and are described for example in Sambrooket al., 1989, Molecular Cloning, 2nd Edition, Cold Spring HarborLaboratory Press, Cold Spring Harbour, N.Y. The hybridization productmay be assayed using techniques known in the art. The nucleotide probemay be labeled with a detectable marker as described herein and thehybridization product may be assayed by detecting the detectable markeror the detectable change produced by the detectable marker.

Primers

The identification of the nucleic acid molecule of the invention alsopermits the identification and isolation, or synthesis of nucleotidesequences which may be used as primers to amplify a polynucleotidemolecule of the invention, for example in polymerase chain reaction(PCR). The length and bases of the primers for use in the PCR areselected so that they will hybridize to different strands of the desiredsequence and at relative positions along the sequence such that anextension product synthesized from one primer when it is separated fromits template can serve as a template for extension of the other primerinto a nucleic acid of defined length. Primers which may be used in theinvention are oligonucleotides i.e. molecules containing two or moredeoxyribonucleotides of the nucleic acid molecule of the invention whichoccur naturally as in a purified restriction endonuclease digest or areproduced synthetically using techniques known in the art such as, forexample, phosphotriester and phosphodiester methods or automatedtechniques (Connolly B. A., 1987, Nucl. Acid Res., 15: 3131-3139). Theprimers are capable of acting as a point of initiation of synthesis whenplaced under conditions which permit the synthesis of a primer extensionproduct which is complementary to the DNA sequence of the invention e.g.in the presence of nucleotide substrates, an agent for polymerizationsuch as DNA polymerase and at suitable temperature and pH. Preferably,the primers are sequences that do not form secondary structures by basepairing with other copies of the primer or sequences that form a hairpin configuration. The primer may be single or double-stranded. When theprimer is double-stranded it may be treated to separate its strandsbefore using it to prepare amplification products. The primer preferablycontains between about 7 and 25 nucleotides.

The primers may be labeled with detectable markers which allow fordetection of the amplified products. Suitable detectable markers areradioactive markers such as ³²P, ³⁵S, ¹²⁵I and ³H, luminescent markerssuch as chemiluminescent markers, preferably luminol and fluorescentmarkers, preferably dansyl chloride, fluorescein-5-isothiocyanate and4-fluor-7-nitrobenz-2-oxa-1,3 diazole and cofactors such as biotin. Itwill be appreciated that the primers may contain non-complementarysequences provided that a sufficient amount of the primer contains asequence which is complementary to a nucleic acid molecule of theinvention or oligonucleotide sequence thereof, which is to be amplified.Restriction site linkers may also be incorporated into the primersallowing for digestion of the amplified products with the appropriaterestriction enzymes facilitating cloning and sequencing of the amplifiedproduct.

Assays—Amplifying Sequences

Thus, a method of determining the presence of a nucleic acid moleculehaving a sequence encoding the subject polynucleotides or apredetermined oligonucleotide fragment thereof in a sample, is providedcomprising treating the sample with primers which are capable ofamplifying the nucleic acid molecule or the predeterminedoligonucleotide fragment thereof in a polymerase chain reaction to formamplified sequences, under conditions which permit the formation ofamplified sequences and, assaying for amplified sequences.

The polymerase chain reaction refers to a process for amplifying atarget nucleic acid sequence as generally described in Innis M. A. andGelfand D. H., 1989, PCR Protocols, A Guide to Methods and Applications,Innis M. A., Gelfand D. H., Shinsky J. J. and White T. J. (eds),Academic Press, NY, pp. 3-12, which are incorporated herein byreference. Conditions for amplifying a nucleic acid template aredescribed in Innis M. A. and Gelfand D. H., #1989, PCR Protocols, AGuide to Methods and Applications, Innis M. A., Gelfand D. H., ShinskyJ. J and White T. J. (eds), Academic Press, NY, pp. 3-12, which is alsoincorporated herein by reference.

The amplified products can be isolated and distinguished based on theirrespective sizes using techniques known in the art. For example, afteramplification, the DNA sample can be separated on an agarose gel andvisualized, after staining with ethidium bromide, under ultraviolet (UV)light. DNA may be amplified to a desired level and a further extensionreaction may be performed to incorporate nucleotide derivatives havingdetectable markers such as radioactive labeled or biotin labelednucleoside triphosphates. The primers may also be labeled withdetectable markers. The detectable markers may be analyzed byrestriction and electrophoretic separation or other techniques known inthe art.

The conditions which may be employed in the methods of the inventionusing PCR are those which permit hybridization and amplificationreactions to proceed in the presence of DNA in a sample and appropriatecomplementary hybridization primers. Conditions suitable for thepolymerase chain reaction are generally known in the art. For example,see Innis M. A. and Gelfand D. H., 1989, PCR Protocols, A Guide toMethods and Applications, Innis M. A., Gelfand D. H., Shinsky J. J. andWhite T. J. (eds), Academic Press, NY, pp. 3-12, which is incorporatedherein by reference. Preferably, the PCR utilizes polymerase obtainedfrom thermophilic bacterium Thermus aquaticus (Taq polymerase, GeneAmpKit, Perkin Elmer Cetus) or other thermostable polymerase may be used toamplify DNA template strands. It will be appreciated that othertechniques such as the Ligase Chain Reaction (LCR) and Nucleic-AcidSequence Based Amplification (NASBA) may be used to amplify a nucleicacid molecule of the invention. In LCR, two primers which hybridizeadjacent to each other on the target strand are ligated in the presenceof the target strand to produce a complementary strand (Backman, 1991and European Published Application No. 0320308, published Jun. 14,1989). NASBA is a continuous amplification method using two primers, oneincorporating a promoter sequence recognized by an RNA polymerase andthe second derived from the complementary sequence of the targetsequence to the first primer (U.S. Pat. No. 5,130,238 to Malek).

Vectors

The present invention also teaches vectors which comprise apolynucleotide or polynucleotides of the present invention, host cellswhich are genetically engineered with vectors of the invention and theproduction of polynucleotides of the invention by recombinanttechniques.

In accordance with this aspect of the invention the vector may be, forexample, a plasmid vector, a single or double-stranded phage vector, ora single or double-stranded RNA or DNA viral vector. In certainembodiments in this regard, the vectors provide for specific expression.Such specific expression may be inducible expression or expression onlyin certain types of cells or both inducible and cell-specific.Particular among inducible vectors are vectors that can be induced forexpression by environmental factors that are easy to manipulate, such astemperature and nutrient additives. A variety of vectors suitable tothis aspect of the invention, including constitutive and inducibleexpression vectors for use in prokaryotic and eukaryotic hosts, are wellknown and employed routinely by those of skill in the art. Such vectorsinclude, among others, chromosomal, episomal and virus-derived vectors,e.g., vectors derived from bacterial plasmids, from bacteriophage, fromtransposons, from yeast episomes, from insertion elements, from yeastchromosomal elements, from viruses such as baculoviruses, papova virusessuch as SV40, vaccinia viruses, adenoviruses, fowl pox viruses,pseudorabies viruses and retroviruses, and vectors derived fromcombinations thereof, such as those derived from plasmid andbacteriophage genetic elements, such as cosmids and phagemids. All ofthese may be used for expression in accordance with this aspect of thepresent invention.

The following vectors, which are commercially available, are provided byway of example. Among vectors for use in bacteria are pQE-9, pQE-16,pQE-30, pQE-40, pQE-50 and pQE-60 (Qiagen); pCR11, pCR11-TOPO, pTrcHisand pBAD-TOPO (Invitrogen); pGEM-3Z, pGEMEX-1, pET-5 (Promega); pBSphagemid vectors, Phagescript vectors, Bluescript vectors, pCAL, pET-3and pSPUTK (Stratagene); pTrc99A, pKzK223-3, pKK232-8 and pRIT2T(Pharmacia); pMAL (New England Biolabs); and pBR322 (ATCC 37017). Amongeukaryotic vectors are pGAPZ, pYES2, pYES2/CT and pcDNA3.1 (Invitrogen);pCAT3 and pGL3 (Promega); pCMV-Script, pXT1, pDual, pCMVLacI, pESC,HybriZAP2.1. ImmunoZAP and pRS (Stratagene); and pSVK3, pSVL and pMSG(Pharmacia). These vectors are listed solely by way of illustration ofthe many commercially available and well known vectors that areavailable to those of skill in the art for use in accordance with thisaspect of the present invention. It will be appreciated that any otherplasmid or vector suitable for, for example, introduction, maintenance,propagation or expression of a polynucleotide or polypeptide of theinvention in a host may be used in this aspect of the invention.Generally, any vector suitable to maintain, propagate or expresspolynucleotides to express a polypeptide or polynucleotide in a host maybe used for expression in this regard. The DNA sequence in theexpression vector is operatively linked to appropriate expressioncontrol sequence(s), including, for instance, a promoter to direct mRNAtranscription. Promoter regions can be selected from any desired geneusing vectors that contain a reporter transcription unit lacking apromoter region, such as a chloramphenicol acetyl transferase (CAT)transcription unit, downstream of restriction site or sites forintroducing a candidate promoter fragment; i.e., a fragment that maycontain a promoter. As is well known, introduction into the vector of apromoter-containing fragment at the restriction site upstream of the CATgene engenders production of CAT activity, which can be detected bystandard CAT assays. Vectors suitable to this end are well known andreadily available, such as pKK232-8 and pCAT3. Promoters for expressionof polynucleotides of the present invention include not only well knownand readily available promoters, but also promoters that readily may beobtained by the foregoing technique, using a reporter gene. Among knownprokaryotic promoters suitable for expression of polynucleotides andpolypeptides in accordance with the present invention are the E. colilacI and lacZ promoters, the T3 and T7 promoters, the gpt promoter, thelambda PR and PL promoters, and the trp promoter. Among knowneulkaryotic promoters suitable in this regard are the CMV immediateearly promoter, the HSV thymidine kinase promoter, the early and lateSV40 promoters, the promoters of retroviral LTRs, such as those of theRous sarcoma virus (RSV), and metallothionein promoters, such as themouse metallothionein-1 promoter.

Vectors for propagation and expression generally will include selectablemarkers and amplification regions, such as, for example, those set forthin Sambrook et al., supra.

Host Cells

As hereinbefore mentioned, the present invention also teaches host cellswhich are genetically engineered with vectors of the invention.

Polynucleotide constructs in host cells can be used in a conventionalmanner to produce the gene product encoded by the recombinant sequence.The subject polynucleotides or polypeptides products or isoforms orparts thereof, may be obtained by expression in a suitable host cellusing techniques known in the art. Suitable host cells includeprokaryotic or eukaryotic organisms or cell lines, for examplebacterial, mammalian, yeast, or other fungi, viral, plant or insectcells. Methods for transforming or transfecting cells to express foreignDNA are well known in the art (See for example, Itakura et al., U.S.Pat. No. 4,704,362; Murray et al., U.S. Pat. No. 4,801,542; McKnight etal., U.S. Pat. No. 4,935,349; Hagen et al., U.S. Pat. No. 4,784,950;Axel et al., U.S. Pat. No. 4,399,216; Goeddal et al., U.S. Pat. No.4,766,075 and Sambrook et al., 1989, Molecular Cloning, 2nd Edition,Cold Spring Harbor Laboratory Press, Cold Spring Harbour, N.Y. all ofwhich are incorporated herein by reference). Representative examples ofappropriate hosts include bacterial cells, such as Streptococci,Staphylococci, E. coli, Streptomyces and Bacillus subtilis; fungalcells, such as yeast cells and Aspergillus cells; insect cells such asDrosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS-1,ZR-75-1, Chang, HeLa, C127, 3T3, HepG2, BHK, 293 and Bowes melanomacells; and plant cells.

Host cells can be genetically engineered to incorporate polynucleotidesand express polynucleotides of the present invention. Introduction ofpolynucleotides into the host cell can be effected by calcium phosphatetransfection, DEAF-dextran mediated transfection, transvection,microinjection, cationic lipid-mediated transfection, electroporation,transduction, scrape loading, ballistic introduction, infection or othermethods. Such methods are described in many standard laboratory manuals,such as Davis et al., 1986, Basic Methods in Molecular Biology,Elsevier, N.Y. and Sambrook et al., 1989, Molecular Cloning, 2ndEdition, Cold Spring Harbor Laboratory Press, Cold Spring Harbour, N.Y.

Production of the Subject Polypeptides

As hereinbefore mentioned, the present invention also teaches theproduction of polynucleotides of the invention by recombinanttechniques.

The subject polynucleotides encode polypeptides which are the matureprotein plus additional amino- or carboxyl-terminal amino acids, oramino acids interior to the mature polypeptide (when the mature form hasmore than one polypeptide chain, for instance). Such sequences may playa role in processing of a protein from precursor to a mature form, mayallow protein transport, may lengthen or shorten protein half-life ormay facilitate manipulation of a protein for assay or production, amongother things. Generally, as is the case in vivo, the additional aminoacids may be processed away from the mature protein by cellular enzymes.A precursor protein, having the mature form of the polypeptide fused toone or more prosequences may be an inactive form of the polypeptide.When prosequences are removed such inactive precursors generally areactivated. Some or all of the prosequences may be removed beforeactivation. Generally, such precursors are called proproteins.

Thus, a polynucleotide of the present invention may encode a matureprotein, a mature protein plus a leader sequence (which may be referredto as a preprotein), a precursor of a mature protein having one or moreprosequences which are not the leader sequences of a preprotein, or apreproprotein, which is a precursor to a proprotein, having a leadersequence and one or more prosequences, which generally are removedduring processing steps that produce active and mature forms of thepolypeptide.

The polypeptides of the invention may be prepared by culturing thehost/vector systems described above, in order to express the recombinantpolypeptides. Recombinantly produced subject protein or parts thereof,may be further purified using techniques known in the art such ascommercially available protein concentration systems, by salting out theprotein followed by dialysis, by affinity chromatography, or using anionor cation exchange resins. Mature proteins can be expressed in mammaliancells, yeast, bacteria, or other cells under the control of appropriatepromoters. Cell-free translation systems can also be employed to producesuch proteins using DNA derived from the DNA constructs of the presentinvention. Appropriate cloning and expression vectors for use withprokaryotic and eukaryotic hosts are described by Sambrook et al.,supra.

Polynucleotides of the invention, encoding the heterologous structuralsequence of a polynucleotide or polypeptide of the invention generallywill be inserted into a vector using standard techniques so that it isoperably linked to the promoter for expression. The polynucleotide willbe positioned so that the transcription start site is locatedappropriately 5′ to a ribosome binding site. The ribosome binding sitewill be 5′ to the AUG that initiates translation of the polynucleotideor polypeptide to be expressed. Generally, there will be no other openreading frames that begin with an initiation codon, usually AUG, and liebetween the ribosome binding site and the initiation codon. Also,generally, there will be a translation stop codon at the end of theexpressed polynucleotide and there will be a polyadenylation signal inconstructs for use in eukaryotic hosts. Transcription termination signalappropriately disposed at the 3′ end of the transcribed region may alsobe included in the polynucleotide construct.

For secretion of the translated protein into the lumen of theendoplasmic reticulum, into the periplasmic space or into theextracellular environment, appropriate secretion signals may beincorporated into the expressed polynucleotide or polypeptide. Thesesignals may be endogenous to the polynucleotide or they may beheterologous signals. Microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents, or other such methods know to those skilled in the art. Asubject polynucleotide or polypeptide can be recovered and purified fromrecombinant cell cultures by known methods including ammonium sulfate orethanol precipitation, acid extraction, anion or cation exchangechromatography, phosphocellulose chromatography, hydrophobic interactionchromatography, affinity chromatography, hydroxylapatite chromatographyand lectin chromatography. Most preferably, high performance liquidchromatography is employed for purification. Well known techniques forrefolding protein may be employed to regenerate active conformation whenthe polynucleotide is denatured during isolation and or purification.

A nucleic acid molecule of the invention may be cloned into aglutathione S-transferase (GST) gene fusion system for example thepGEX-1T, pGEX-2T and pGEX-3× of Pharmacia. The fused gene may contain astrong lac promoter, inducible to a high level of expression by IPTG, asa regulatory element. Thrombin or factor Xa cleavage sites may bepresent which allow proteolytic cleavage of the desired polypeptide fromthe fusion product. The glutathione S-transferase-subject polypeptidefusion protein may be easily purified using a glutathione sepharose 4Bcolumn, for example from Pharmacia. The 26 kDa glutathione S-transferasepolypeptide can be cleaved by thrombin (PGEX-1T or pGEX-2T) or factor Xa(pGEX-3×) and resolved from the polypeptide using the same affinitycolumn. Additional chromatographic steps can be included if necessary,for example Sephadex or DEAE cellulose. The two enzymes may be monitoredby protein and enzymatic assays and purity may be confirmed usingSDS-PAGE.

The subject protein or parts thereof may also be prepared by chemicalsynthesis using techniques well known in the chemistry of proteins suchas solid phase synthesis (Merrifield, 1964, J. Am. Chem. Assoc., 85:2149-2154) or synthesis in homogenous solution (Houbenweyl et al., 1987,Methods of Organic Chemistry, Wansch E. (ed), Vol. 15 I and II, Thieme,Germany).

Within the context of the present invention, the subject polypeptideincludes various structural forms of the primary protein which retainbiological activity. For example, the subject polypeptide may be in theform of acidic or basic salts or in neutral form. In addition,individual amino acid residues may be modified by oxidation orreduction. Furthermore, various substitutions, deletions or additionsmay be made to the amino acid or nucleic acid sequences, the net effectbeing that biological activity of the subject polypeptide is retained.Due to code degeneracy, for example, there may be considerable variationin nucleotide sequences encoding the same amino acid.

The polypeptide may be expressed in a modified form, such as a fusionprotein, and may include not only secretion signals but also additionalheterologous functional regions. Thus. for instance, a region ofadditional amino acids, particularly charged amino acids, may be addedto the carboxyl- or amino-terminus of the polypeptide to improvestability and persistence in the host cell during purification or duringsubsequent handling and storage. Also, fusion proteins may be added tothe polynucleotide or polypeptide to facilitate purification. Suchregions may be removed prior to final preparation of the polynucleotideor polypeptide. The addition of peptide moieties to polynucleotides orpolypeptides to engender secretion or excretion, to improve stability orto facilitate purification, among others, are familiar and routinetechniques in the art. In drug discovery, for example, proteins havebeen fused with antibody Fc portions for the purpose of high-throughputscreening assays to identify antagonists (Bennett et al., 1995, J. Mol.Recognit., 8: 52-58, and Johanson et al., 1995, J. Biol. Chem., 270:9459-9471).

Antibodies

With respect to protein-based testing, antibodies can be generated tothe elongase gene product using standard immunological techniques,fusion proteins or synthetic peptides as described herein. Monoclonalantibodies can also be produced using now conventional techniques suchas those described in Waldmann T. A., 1991, Science, 252: 1657-1662 andHarlow E. and Lane D. (eds.), 1988, Antibodies: A Laboratory Manual,Cold Harbour Press. Cold Harbour, N.Y. It will also be appreciated thatantibody fragments, i.e. Fab′ fragments, can be similarly employed.Immunoassays, for example ELISAs, in which the test sample is contactedwith antibody and binding to the gene product detected, can provide aquick and efficient method of determining the presence and quantity ofthe elongase gene product. For example, the antibodies can be used totest the effect of pharmaceuticals in subjects enrolled in clinicaltrials.

Thus, the present invention also provides polyclonal and/or monoclonalantibodies and fragments thereof, and immunologic binding equivalentsthereof, which are capable of specifically binding to the subjectpolypeptides and fragments thereof or to polynucleotide sequences fromthe subject polynucleotide region, particularly from the subjectpolypeptide locus or a portion thereof. The term “antibody” is used bothto refer to a homogeneous molecular entity, or a mixture such as a serumproduct made up of a plurality of different molecular entities.Polypeptides may be prepared synthetically in a peptide synthesizer andcoupled to a carrier molecule (e.g., keyhole limpet hemocyanin) andinjected over several months into rabbits. Rabbit sera is tested forimmunoreactivity to the subject polypeptide or fragment. Monoclonalantibodies may be made by injecting mice with the protein polypeptides,fusion proteins or fragments thereof. Monoclonal antibodies are screenedby ELISA and tested for specific immunoreactivity with subjectpolypeptide or fragments thereof (Harlow E. and Lane D. (eds.), 1988,Antibodies. A Laboratory Manual, Cold Harbour Press, Cold Harbour,N.Y.). These antibodies are useful in assays as well as pharmaceuticals.

Once a sufficient quantity of desired polypeptide has been obtained, itmay be used for various purposes. A typical use is the production ofantibodies specific for binding. These antibodies may be eitherpolyclonal or monoclonal, and may be produced by in vitro or in vivotechniques well known in the art. For production of polyclonalantibodies, an appropriate target immune system, typically mouse orrabbit, is selected. Substantially purified antigen is presented to theimmune system in a fashion determined by methods appropriate for theanimal and by other parameters well known to immunologists. Typicalroutes for injection are in footpads, intramuscularly,intraperitoneally, or intradermally. Of course, other species may besubstituted for mouse or rabbit. Polyclonal antibodies are then purifiedusing techniques known in the art, adjusted for the desired specificity.

An immunological response is usually assayed with an immunoassay.Normally, such immunoassays involve some purification of a source ofantigen, for example, that produced by the same cells and in the samefashion as the antigen. A variety of immunoassay methods are well knownin the art, such as in Harlow E. and Lane D. (eds.), 1988, Antibodies. ALaboratory Manual, Cold Harbour Press, Cold Harbour, N.Y., or Goding J.W., 1996, Monoclonal Antibodies. Principles and Practice. Production andApplication of Monoclonal Antibodies in Cell Biology, Biochemistry andImmunology, 3^(rd) edition, Academic Press, NY.

Monoclonal antibodies with affinities of 10⁸ M⁻¹ or preferably 10⁹ to10¹⁰M⁻¹ or stronger will typically be made by standard procedures asdescribed in Harlow E. and Lane D. (eds.), 1988, Antibodies. ALaboratory Manual, Cold Harbour Press, Cold Harbour, N.Y. or Coding J.W., 1996, Monoclonal Antibodies. Principles and Practice. Production andApplication of Monoclonal Antibodies in Cell Biology, Biochemistry andImmunology, 3^(rd) edition, Academic Press, NY. Briefly, appropriateanimals will be selected and the desired immunization protocol followed.After the appropriate period of time, the spleens of such animals areexcised and individual spleen cells fused, typically, to immortalizedmyeloma cells under appropriate selection conditions. Thereafter, thecells are clonally separated and the supernatants of each clone testedfor their production of an appropriate antibody specific for the desiredregion of the antigen.

Other suitable techniques involve in vitro exposure of lymphocytes tothe antigenic polypeptides, or alternatively, to selection of librariesof antibodies in phage or similar vectors (Huse et al., 1989, Science,246: 1275-1281). The polypeptides and antibodies of the presentinvention may be used with or without modification. Frequently,polypeptides and antibodies will be labeled by joining, eithercovalently or non-covalently, a substance which provides for adetectable signal. A wide variety of labels and conjugation techniquesare known and are reported extensively in both the scientific and patentliterature. Suitable labels include radionuclides, enzymes, substrates,cofactors, inhibitors, fluorescent agents, chemiluminescent agents,magnetic particles and the like. Patents teaching the use of such labelsinclude U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345;4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulinsmay be produced (see U.S. Pat. No. 4,816,567).

Generation of polyclonal Antibody Against the Subject Polynucleotide

Sequences of the subject polynucleotide coding sequence are expressed asfusion protein in E. coli. The overexpressed protein is purified by gelelution and used to immunize rabbits and mice using a procedure similarto the one described by Harlow E. and Lane D. (eds.), 1988, Antibodies:A Laboratory Manual, Cold Harbour Press, Cold Harbour, N.Y. Thisprocedure has been shown to generate antibodies against various otherproteins (for example, see Kraemer et al., 1993, J. Lipid Res., 34:663-671).

Briefly, a stretch of coding sequence selected from the subjectpolynucleotide is cloned as a fusion protein in plasmid pET5A (Novagen,Wis.) or pMAL system (New England Biolabs, U.S.). After induction withIPTG, the overexpression of a fusion protein with the expected molecularweight is verified by SDS-PAGE. Fusion protein is purified from the gelby electroelution. The identification of the protein as the subjectpolypeptide fusion product can be verified by protein sequencing at theN-terminus. Next, the purified protein is used as immunogen in rabbits.Rabbits are immunized with 100 μg of the protein in complete Freund'sadjuvant and boosted twice in 3 week intervals, first with 100 μg ofimmunogen in incomplete Freund's adjuvant followed by 100 μg ofimmunogen in PBS. Antibody containing serum is collected two weeksthereafter.

This procedure is repeated to generate antibodies against the mutantforms of the subject polypeptide. These antibodies, in conjunction withantibodies to wild type subject polypeptide, are used to detect thepresence and the relative level of the mutant forms in various tissuesand biological fluids.

Generation of Monoclonal Antibodies Specific for the Subject Polypeptide

Monoclonal antibodies are generated according to the following protocol.Mice are immunized with immunogen comprising intact subject polypeptideor its peptides (wild type or mutant) conjugated to keyhole limpethemocyanin using glutaraldehyde or EDC as is well known.

The immunogen is mixed with an adjuvant. Each mouse receives fourinjections of 10 to 100 μg of immunogen and after the fourth injectionblood samples are taken from the mice to determine if the serum containsantibody to the immunogen. Serum titer is determined by ELISA or RIA.Mice with sera indicating the presence of antibody to the immunogen areselected for hybridoma production.

Spleens are removed from immune mice and a single cell suspension isprepared as described by Harlow E. and Lane D. (eds.), 1988, Antibodies.A Laboratory Manual, Cold Harbour Press, Cold Harbour, N.Y. Cell fusionsare performed essentially as described by Kohler G. and Milstein C.,1975, Nature, 256: 495-497. Briefly, P3.65.3 myeloma cells (AmericanType Culture Collection, Rockville, Md.) are fused with immune spleencells using polyethylene glycol as described by Harlow E. and Lane D.(eds.), 1988, Antibodies: A Laboratory Manual, Cold Harbour Press, ColdHarbour, N.Y. Cells are plated at a density of 2×10⁵ cells/well in 96well tissue culture plates. Individual wells are examined for growth andthe supernatants of wells with growth are tested for the presence ofsubject polypeptide specific antibodies by ELISA or RIA using wild typeor mutant target protein. Cells in positive wells are expanded andsubcloned to establish and confirm monoclonality. Clones with thedesired specificities are expanded and grown as ascites in mice or in ahollow fiber system to produce sufficient quantities of antibody forcharacterization and assay development.

Sandwich Assay for the Subject Polypeptide

Monoclonal antibody is attached to a solid surface such as a plate,tube, bead, or particle. Preferably, the antibody is attached to thewell surface of a 96-well ELISA plate. A 100 μl sample (e.g., serum,urine, tissue cytosol) containing the subject polypeptide/protein(wild-type or mutant) is added to the solid phase antibody. The sampleis incubated for 2 hrs at room temperature. Next the sample fluid isdecanted, and the solid phase is washed with buffer to remove unboundmaterial. One hundred μl of a second monoclonal antibody (to a differentdeterminant on the subject polypeptide/protein) is added to the solidphase. This antibody is labeled with a detector molecule or atom (e.g.,¹²⁵I, enzyme, fluorophore, or a chromophore) and the solid phase withthe second antibody is incubated for two hrs at room temperature. Thesecond antibody is decanted and the solid phase is washed with buffer toremove unbound material.

The amount of bound label, which is proportional to the amount ofsubject polypeptide/protein present in the sample, is quantitated.Separate assays are performed using monoclonal antibodies which arespecific for the wild-type subject polypeptide as well as monoclonalantibodies specific for each of the mutations identified in subjectpolypeptide.

Detecting Presence of or Predisposition for Disorders Affected by LipidMetabolism and Monitoring Treatment of Same

As previously discussed, lipid metabolism is frequently disregulated indisease. It is likely that genetic polymorphisms in elongase genes willcontribute to disease susceptibility. The subject polynucleotides taughtherein are useful to detect genetic polymorphisms of the subjectpolynucleotides, or to detecting changes in the level of expression ofthe subject polynucleotides, as a diagnostic tool. Detection of anaberrant form of the subject polynucleotide, or a decrease or increasein the level of expression of the subject polynucleotide in a eukaryote,particularly a mammal, and especially a human, will provide a method fordiagnosis of a disease. Eukaryotes (herein also “individual(s)”),particularly mammals, and especially humans, exhibiting geneticpolymorphisms of the subject polynucleotides, or changes in expressionof the subject polynucleotides may be detected by a variety oftechniques.

Since elongase genes are widely expressed, test samples of the subjectcan be obtained from a variety of tissues including blood. An elongasegene test can also be included in panels of prenatal tests sinceelongase genes, DNA, RNA or protein can also be assessed in amnioticfluid. Quantitative testing for elongase gene transcript and geneproduct is thus also contemplated within the scope of the presentinvention.

Nucleic acid and protein-based methods for screening geneticpolymorphisms in elongase genes are all within the scope of the presentteachings. For example, knowing the sequence of the elongase gene, DNAor RNA probes can be constructed and used to detect mutations inelongase genes through hybridization with genomic DNA in a tissue suchas blood using conventional techniques. RNA or cDNA probes can besimilarly probed to screen for mutations in elongase genes or forquantitative changes in expression. A mixture of different probes, i.e.“probe cocktail”, can also be employed to test for more than onemutation. With respect to nucleic acid-based testing, genomic DNA may beused directly for detection of a specific sequence or may be amplifiedenzymatically in vitro by using PCR prior to analysis (Saiki et al.,1985, Science, 230: 1350-1353 and Saiki et al., 1986, Nature, 324:163-166). Reviews of this subject have been presented by Caskey C. T.,1989, Science, 236: 1223-1229 and by Landegren et al., 1989, Science,242: 229-237. The detection of specific DNA sequence may be achieved bymethods such as hybridization using specific oligonucleotides (Wallaceet al., 1986, Cold Spring Harbour Symp. Quant. Biol., 51: 257-261),direct DNA sequencing (Church et al., 1988, Proc. Natl. Acad. Sci., 81:1991-1995, the use of restriction enzymes (Flavell et al., 1978, Cell,15: 25-41; Geever et al., 1981, Proc. Natl. Acad. Sci., 78: 5081-5085),discrimination on the basis of electrophoretic mobility in gels withdenaturing reagent (Myers et al., 1986, Cold Spring Harbour Sym. Quant.Biol., 51: 275-284), RNase protection (Myers et al., 1985, Science, 230:1242-1246), chemical cleavage (Cotton et al., 1985, Proc. Natl. Acad.Sci., 85: 4397-4401), and the ligase-mediated detection procedure(Landegren et al., 1988, Science, 241: 1077-1080). Using PCR,characterization of the level of or condition of the subjectpolynucleotides present in the individual may be made by comparativeanalysis.

With respect to protein-based testing, antibodies can be generated tothe elongase gene product using standard immunological techniques,fusion proteins or synthetic peptides as described herein.

With the characterization of the elongase gene product and its function,functional assays can also be used for elongase gene diagnosis andscreening and to monitor treatment. For example, enzymatic testing todetermine levels of gene function, rather than direct screening of theelongase gene or product, can be employed. Testing of this nature hasbeen utilized in other diseases and conditions, such as in Tay-Sachs.

The invention thus provides a process for detecting disease by usingmethods known in the art and methods described herein to detect changesin expression of or mutations to the subject polynucleotides. Forexample, decreased expression of a subject polynucleotide can bemeasured using any one of the methods well known in the art for thequantification of polynucleotides, such as, for example, PCR, RT-PCR,DNase protection, Northern blotting and other hybridization methods.Thus, the present invention provides a method for detecting disordersaffected by lipid metabolism, and a method for detecting a geneticpre-disposition for such diseases including eczema, cardiovasculardisorders (including but not limited to hypertriglyceridemia,dyslipidemia, atherosclerosis, coronary artery disease, cerebrovasculardisease and peripheral vascular disease), inflammation (including butnot limited to sinusitis, asthma, pancreatitis, osteoarthritis,rheumatoid arthritis and acne), body weight disorders (including but notlimited to obesity, cachexia and anorexia), psychiatric disorders,cancer, cystic fibrosis, pre-menstrual syndrome, diabetes and diabeticcomplications.

Drug Screening Assays

The present teachings provide methods for screening compounds toidentify those which enhance (agonist) or block (antagonist) the actionof subject polypeptides or polynucleotides, such as its interaction withfatty acid binding molecules. The identification of the subjectpolynucleotides in inherited fatty acid disorders, combined withadvances in the field of transgenic methods, provides the informationnecessary to further study human diseases. This is extraordinarilyuseful in modeling familial forms of fatty acid disorders and otherdiseases of fatty acid metabolism including eczema, cardiovasculardisorders (including but not limited to hypertriglyceridemia,dyslipidemia, atherosclerosis, coronary artery disease, cerebrovasculardisease and peripheral vascular disease), inflammation (including butnot limited to sinusitis, asthma, pancreatitis, osteoarthritis,rheumatoid arthritis and acne), body weight disorders (including but notlimited to obesity, cachexia and anorexia), psychiatric disorders,cancer, cystic fibrosis, pre-menstrual syndrome, diabetes and diabeticcomplications. Drug screening assays are made effective by use of thecontrol regions of the genes described in the present invention or partof it, in a yeast based DNA-protein interaction assay (yeastone-hybrid). The use of the genes described here, or parts thereof, orthe transcribed RNA in a yeast protein-protein interaction (2-hybrid) orprotein-RNA interaction assays for drug screening also provide effectivedrug screening methods. Such interacting molecules can also bereconstructed in vitro for drug screening purposes. For example, toscreen for agonists or antagonists, a synthetic reaction mix, a cellularcompartment, such as a membrane, cell envelope or cell wall, or apreparation of any thereof, may be prepared from a cell that expresses amolecule that binds a subject polynucleotide. The preparation isincubated with labeled polynucleotide in the absence or the presence ofa candidate molecule which may be an agonist or antagonist. The abilityof the candidate molecule to bind the binding molecule is reflected indecreased binding of the labeled ligand. Effects of potential agonistsand antagonists may by measured, for instance, by determining activityof a reporter system following interaction of the candidate moleculewith a cell or appropriate cell preparation, and comparing the effect toa baseline (control) measurement. Reporter systems that may be useful inthis regard include, but are not limited to, calorimetric labeledsubstrate converted into product, a reporter gene that is responsive tochanges in elongase enzyme activity, and binding assays known in theart.

Another example of an assay for antagonists is a competitive assay thatcombines a subject polypeptide and a potential antagonist withmembrane-bound subject polypeptide-binding molecules, recombinantsubject polypeptide binding molecules, natural substrates or ligands, orsubstrate or ligand mimetics, under appropriate conditions for acompetitive inhibition assay. A subject polypeptide can be labeled, suchas by radioactivity or a calorimetric compound, such that the number ofsubject polypeptide molecules bound to a binding molecule or convertedto product can be determined accurately to assess the effectiveness ofthe potential antagonist.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential antagonists also may be small organic molecules, peptides,polypeptides, such as closely related proteins or antibodies that bindthe same sites on a binding molecule, without inducing subjectpolypeptide-induced activities, thereby preventing the action of thesubject polypeptide by excluding the subject polypeptide from binding.Potential antagonists include antisense molecules (Okano et al., 1988,EMBO J., 7: 3407-3412). Potential antagonists include compounds relatedto and derivatives of the subject polypeptides.

Potential antagonists include small organic molecules, peptides,polypeptides and antibodies that bind to a polynucleotide or polypeptideof the invention and thereby inhibit or extinguish its activity.Potential agonists may be selected from the group consisting of smallorganic molecules, peptides, polypeptides, antisense molecules,oligonucleotides, polynucleotides, fatty acids, and chemical andfunctional derivatives thereof.

Developing modulators of the biological activities of specific elongasesrequires differentiating elongase isozymes present in a particular assaypreparation. The classical enzymological approach of isolating elongasesfrom natural tissue sources and studying each new isozyme may be used.Another approach has been to identify assay conditions which might favorthe contribution of one isozyme and minimize the contribution of othersin a preparation. Still another approach is the separation of elongasesby immunological means. Each of the foregoing approaches fordifferentiating elongase isozymes is time consuming. As a result manyattempts to develop selective elongase modulators have been performedwith preparations containing more than one isozyme. Moreover, elongasepreparations from natural tissue sources are susceptible to limitedproteolysis and may contain mixtures of active proteolytic products thathave different kinetic, regulatory and physiological properties than thefull length elongases.

Recombinant subject polypeptide products of the invention greatlyfacilitate the development of new and specific modulators. The need forpurification of an isozyme can be avoided by expressing it recombinantlyin a host cell that lacks endogenous elongase activity. Once a compoundthat modulates the activity of the elongase is discovered, itsselectivity can be evaluated by comparing its activity on the particularsubject enzyme to its activity on other elongase isozymes. Thus, thecombination of the recombinant subject polypeptide products of theinvention with other recombinant elongase products in a series ofindependent assays provides a system for developing selective modulatorsof particular elongases. Selective modulators may include, for example,antibodies and other proteins or peptides which specifically bind to thesubject polypeptide or polynucleotide, oligonucleotides whichspecifically bind to the subject polypeptide (see Patent CooperationTreaty International Publication No. WO 93/05182 which describes methodsfor selecting oligonucleotides which selectively bind to targetbiomolecules) or the subject polynucleotide (e.g., antisenseoligonucleotides) and other non-peptide natural or synthetic compoundswhich specifically bind to the subject polynucleotide or polypeptide.Mutant forms of the subject polynucleotide which alter the enzymaticactivity of the subject polypeptide or its localization in a cell arealso contemplated. Crystallization of recombinant subject polypeptidesalone and bound to a modulator, analysis of atomic structure by X-raycrystallography, and computer modeling of those structures are methodsuseful for designing and optimizing non-peptide selective modulators.See, for example, Erickson et al., 1992, Ann. Rep. Med. Chem., 27:271-289 for a general review of structure-based drug design.

Targets for the development of selective modulators include, forexample: (1) the regions of the subject elongases which contact otherproteins and/or localize the proteins within a cell, (2) the regions ofthe proteins which bind substrate, and (3) the phosphorylation site(s)of the subject polypeptides.

Thus, the present invention provides methods for screening and selectingcompounds which promote disorders affected by lipids. As well, thepresent invention provides methods for screening and selecting compoundswhich treat or inhibit progression of diseases associated with lipidmetabolism, such as eczema, cardiovascular disorders (including but notlimited to hypertriglyceridemia, dyslipidemia, atherosclerosis, coronaryartery disease, cerebrovascular disease and peripheral vasculardisease), inflammation (including but not limited to sinusitis. asthma,pancreatitis, osteoarthritis, rheumatoid arthritis and acne), bodyweight disorders (including but not limited to obesity, cachexia andanorexia), psychiatric disorders, cancer, cystic fibrosis, pre-menstrualsyndrome, diabetes and diabetic complications, and other diseases notnecessary related to lipid metabolism.

Protein Interaction Assays for DNA Control Regions, CDS and RNA ofElongase Genes.

Protein interaction is implicated in virtually every biological processin the cell, for example, metabolism, transport, signaling and disease.Development of the yeast 2-hybrid and 1-hybrid systems have made itpossible to study and identify protein-protein interaction, protein-DNAinteraction or protein-RNA interaction in vivo (Fields S, and Song O.,1989, Nature, 340: 245-246; Ulmasov et al., 1997, Science, 276:1865-1868; Furuyama K. and Sassa S., 2000, J. Clin. Invest., 105:757-764 and Gyuris et al., 1993, Cell, 75: 791-803). Because theseinteractions are key to cellular functions, identification ofinteracting partners is the first step towards elucidation of functionand involvement in pathogenesis. New chemical entities that modulate(inhibit or activate) such interactions may have strong pharmaceuticaland therapeutic benefit in human, animal as well as plant diseases. Itis now known that in sideroblastic anemic patients, the interactionbetween succinyl-CoA synthetase and the heme biosynthetic enzymeug-aminolevulinate synthase-E (ALAS-E) is disrupted (Furuyama K. andSassa S., 2000, J. Clin. Invest., 105: 757-764). Inhibition of geneexpression in human cells through small molecule-RNA interaction havebeen recently described (Hwang et al., 1999, Proc. Natl. Acad. Sci., 96:12997-13002). The use of protein-RNA inhibition technology is apotential approach for development of anti-HIV therapeutics (Hamy etal., 1997, Proc. Natl. Acad. Sci., 94: 3548-3553 and Mei et al., 1998,Biochemistry, 37: 14204-14212).

Drug Design

Antagonists and agonists and other compounds of the present inventionmay be employed alone or in conjunction with other compounds, such astherapeutic compounds. The pharmaceutical compositions may beadministered in any effective, convenient manner, including, forinstance, administration by direct microinjection into the affectedarea, or by intravenous or other routes. These compositions of thepresent invention may be employed in combination with a non-sterile orsterile carrier or carriers for use with cells, tissues or organisms,such as a pharmaceutical carrier suitable for administration to asubject. Such compositions comprise, for instance, a medium additive ora therapeutically effective amount of antagonists or agonists of theinvention and a pharmaceutically acceptable carrier or excipient. Suchcarriers may include, but are not limited to, saline, buffered saline,dextrose, water, glycerol, ethanol and combinations thereof. Theformulation is prepared to suit the mode of administration.

Modulation of elongase gene function can be accomplished by the use oftherapeutic agents or drugs which can be designed to interact withdifferent aspects of elongase structure or function. For example, a drugor antibody can bind to a structural fold of the protein to correct adefective structure. Alternatively, a drug might bind to a specificfunctional residue and increase its affinity for a substrate orcofactor. Efficacy of a drug or agent can be identified by a screeningprogram in which modulation is monitored in vitro in cell systems inwhich a defective elongase is expressed.

Alternatively, drugs can be designed to modulate the activity ofproteins of elongase genes from knowledge of the structure and functioncorrelations for such proteins and from knowledge of the specific defectin various mutant proteins (Copsey et al., 1988, Genetically EngineeredHuman Therapeutic Drugs, Stockton Press, NY).

Gene Therapy

A variety of gene therapy approaches may be used in accordance with theinvention to modulate expression of the subject polynucleotides in vivo.For example, antisense DNA molecules may be engineered and used to blocktranslation of mRNA of the subject polynucleotides in vivo.Alternatively, ribozyme molecules may be designed to cleave and destroythe mRNA of the subject polynucleotides in vivo. In another alternative,oligonucleotides designed to hybridize to the 5′ region of the subjectpolynucleotide (including the region upstream of the coding sequence)and form triple helix structures may be used to block or reducetranscription of the subject polynucleotide. In yet another alternative,nucleic acid encoding the full length wild-type subject polynucleotidemay be introduced in vivo into cells which otherwise would be unable toproduce the wild-type subject polynucleotide product in sufficientquantities or at all.

For example, in conventional replacement therapy, gene product or itsfunctional equivalent is provided to the patient in therapeuticallyeffective amounts. Elongases can be purified using conventionaltechniques such as those described in Deutcher M. (ed.), 1990, Guide toProtein Purification, Meth. Enzymol., Vol. 182. Sufficient amounts ofgene product or protein for treatment can be obtained, for example,through cultured cell systems or synthetic manufacture. Drug therapieswhich stimulate or replace the gene product can also be employed.Delivery vehicles and schemes can be specifically tailored to theparticular target gene.

Gene therapy using recombinant technology to deliver the gene into thepatient's cells, or vectors which will supply the patient with geneproduct in vivo, is also within the scope of the invention. Retroviruseshave been considered preferred vectors for experiments in somatic genetherapy, with a high efficiency of infection and stable integration andexpression (Orkin et al., 1988, Prog. Med. Genet., 7: 130-142). Forexample, elongase cDNAs can be cloned into a retroviral vector anddriven from either its endogenous promoter or from the retroviral LTR(long terminal repeat). Other delivery systems which can be utilizedinclude adeno-associated virus (McLaughlin et al., 1988, J. Virol., 62:1963-1973), vaccinia virus (Moss et al., 1987, Annu. Rev. Immunol., 5:305-324), bovine papilloma virus (Rasmussen et al., 1987, Meth.Enzymol., 139: 642-654), or a member of the herpes virus group such asEpstein-Barr virus (Margolskee et al., 1988, Mol. Cell. Biol., 8:2837-2847).

Antisense, ribozyme and triple helix nucleotides are designed to inhibitthe translation or transcription of the subject polynucleotides. Toaccomplish this, the oligonucleotides used should be designed on thebasis of relevant sequences unique to the subject polynucleotides. Forexample, and not by way of limitation, the oligonucleotides should notfall within those regions where the nucleotide sequence of a subjectpolynucleotide is most homologous to that of other polynucleotides,herein referred to as “unique regions”.

In the case of antisense molecules, it is preferred that the sequence bechosen from the unique regions. It is also preferred that the sequencebe at least 18 nucleotides in length in order to achieve sufficientlystrong annealing to the target mRNA sequence to prevent translation ofthe sequence (Izant J. G and Weintraub H., 1984, Cell, 36: 1007-1015 andRosenberg et al., 1985, Nature, 313: 703-706).

In the case of the “hammerhead” type of ribozymes, it is also preferredthat the target sequences of the ribozymes be chosen from the uniqueregions. Ribozymes are RNA molecules which possess highly specificendoribonuclease activity. Hammerhead ribozymes comprise a hybridizingregion which is complementary in nucleotide sequence to at least part ofthe target RNA, and a catalytic region which is adapted to cleave thetarget RNA. The hybridizing region contains 9 or more nucleotides.Therefore, the hammerhead ribozymes of have a hybridizing region whichis complementary to the sequences listed above and is at least ninenucleotides in length. The construction and production of such ribozymesare well known in the art and are described more fully in Haseloff J.and Gerlach W. L., 1988, Nature, 334: 585-591.

The ribozymes also include RNA endoribonucleases (hereinafter “Cech-typeribozymes”) such as the one which occurs naturally in Tetrahymenathermophila (known as the IVS, or L-19 IVS RNA) and which has beenextensively described by Thomas Cech and collaborators (Zaug et al.,1984, Science, 224: 574-578; Zaug A. J. and Cech T. R. 1986, Science,231: 470-475; Zaug et al., 1986, Nature, 324: 429-433; PatentPublication Treaty International Patent Application No. WO 88/04300 andBeen M. D. and Cech T. R., 1986, Cell, 47: 207-216). The Cechendoribonucleases have an eight base pair active site which hybridizesto a target RNA sequence whereafter cleavage of the target RNA takesplace. Cech-type ribozymes target eight base-pair active site sequencesare present in a subject polynucleotide but not other polynucleotidesfor elongases.

The compounds can be administered by a variety of methods which areknown in the art, including, but not limited to the use of liposomes asa delivery vehicle. Naked DNA or RNA molecules may also be used wherethey are in a form which is resistant to degradation, such as bymodification of the ends, by the formation of circular molecules, or bythe use of alternate bonds including phosphothionate and thiophosphorylmodified bonds. In addition, the delivery of nucleic acid may be byfacilitated transport where the nucleic acid molecules are conjugated topolylysine or transferrin. Nucleic acid may also be transported intocells by any of the various viral carriers, including but not limitedto, retrovirus, vaccinia, adeno-associated virus, and adenovirus.

Alternatively, a recombinant nucleic acid molecule which encodes, or is,such antisense, ribozyme, triple helix, or subject polynucleotidemolecule can be constructed. This nucleic acid molecule may be eitherRNA or DNA. If the nucleic acid encodes an RNA, it is preferred that thesequence be operatively attached to a regulatory element so thatsufficient copies of the desired RNA product are produced. Theregulatory element may permit either constitutive or regulatedtranscription of the sequence. A transfer vector such as a bacterialplasmid or viral RNA or DNA, encoding one or more of the RNAs, may betransfected into cells or cells of an organism (Llewellyn et al., 1987,J. Mol. Biol., 195: 115-123 and Hanahan et al., 1983, J. Mol. Biol.,166: 557-580). Once inside the cell, the transfer vector may replicate,and be transcribed by cellular polymerases to produce the RNA or it maybe integrated into the genome of the host cell. Alternatively, atransfer vector containing sequences encoding one or more of the RNAsmay be transfected into cells or introduced into cells by way ofmicromanipulation techniques such as microinjection, such that thetransfer vector or a part thereof becomes integrated into the genome ofthe host cell.

Composition, Formulation, and Administration of PharmaceuticalCompositions

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is itself known, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping or lyophilizing processes.Pharmaceutical compositions for use in accordance with the presentinvention thus may be formulated in conventional manner using one ormore physiologically acceptable carriers comprising excipients andauxiliaries which facilitate processing of the active compounds intopreparations which can be used pharmaceutically. Proper formulation isdependent upon the route of administration chosen.

For injection, the agents of the invention may be formulated in aqueoussolutions, preferably in physiologically compatible buffers such asHanks' solution, Ringer's solution, or physiological saline buffer. Fortransmucosal administration, penetrants appropriate to the barrier to bepermeated are used in the formulation. Such penetrants are generallyknown in the art.

For oral administration, the compounds can be formulated readily bycombining the active compounds with pharmaceutically acceptable carrierswell known in the art. Such carriers enable the compounds of theinvention to be formulated as tablets, pills, dragees, capsules,liquids, gels, syrups, slurries, suspensions and the like, for oralingestion by a patient to be treated. Pharmaceutical preparations fororal use can be obtained by solid excipient, optionally grinding aresulting mixture, and processing the mixture of granules, after addingsuitable auxiliaries, if desired, to obtain tablets or dragee cores.Suitable excipients are, in particular, fillers such as sugars,including lactose, sucrose, mannitol, or sorbitol, or cellulosepreparations such as, maize starch, wheat starch, rice starch, potatostarch, gelatin, gum tragacanth, methyl cellulose,hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/orpolyvinylpyrrolidone. If desired. disintegrating agents may be added,such as the cross-linked polyvinylpyrrolidone, agar, or alginic acid ora salt thereof such as sodium alginate.

Dragee cores are provided with suitable coatings. For this purpose,concentrated sugar solutions may be used, which may optionally containgum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethyleneglycol, and/or titanium dioxide, lacquer solutions, and suitable organicsolvents or solvent mixtures. Dyestuffs or pigments may be added to thetablets or dragee coatings for identification or to characterizedifferent combinations of active compound doses.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a plasticizer, such as glycerol or sorbitol. The push-fitcapsules can contain the active ingredients in admixture with fillersuch as lactose, binders such as starches, and/or lubricants such astalc or magnesium stearate and, optionally, stabilizers. In softcapsules, the active compounds may be dissolved or suspended in suitableliquids, such as fatty oils, liquid paraffin, or liquid polyethyleneglycols. In addition, stabilizers may be added. All formulations fororal administration should be in dosages suitable for suchadministration.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges (e.g. gelatin) for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multidose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Pharmaceutical formulations for parenteral administration includeaqueous solutions of the active compounds in water-soluble form.Additionally, suspensions of the active compounds may be prepared asappropriate oily injection suspensions. Suitable lipophilic solvents orvehicles include fatty oils such as sesame oil, or synthetic fatty acidesters, such as ethyl oleate or triglycerides, or liposomes. Aqueousinjection suspensions may contain substances which increase theviscosity of the suspension, such as sodium carboxymethyl cellulose,sorbitol, or dextran. Optionally, the suspension may also containsuitable stabilizers or agents which increase the solubility of thecompounds to allow for the preparation of highly concentrated solutions.

Alternatively, the active ingredient may be in powder form forconstitution with a suitable vehicle, e.g., sterile pyrogen-free water,before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt.

A pharmaceutical carrier for the hydrophobic compounds of the inventionis a co-solvent system comprising benzyl alcohol, a nonpolar surfactant,a water-miscible organic polymer, and an aqueous phase. Naturally, theproportions of a co-solvent system may be varied considerably withoutdestroying its solubility and toxicity characteristics. Furthermore, theidentity of the co-solvent components may be varied.

Alternatively, other delivery systems for hydrophobic pharmaceuticalcompounds may be employed. Liposomes and emulsions are well knownexamples of delivery vehicles or carriers for hydrophobic drugs. Certainorganic solvents such as dimethylsulfoxide also may be employed,although usually at the cost of greater toxicity. Additionally, thecompounds may be delivered using a sustained-release system, such assemipermeable matrices of solid hydrophobic polymers containingtherapeutic agent. Various sustained-release materials have beenestablished and are well known by those skilled in the art.Sustained-release capsules may, depending on their chemical nature,release the compounds for a few weeks up to over 100 days. Depending onthe chemical nature and the biological stability of therapeutic reagent,additional strategies for protein stabilization may be employed.

The pharmaceutical compositions also may comprise suitable solid or gelphase carriers or excipients. Examples of such carriers or excipientsinclude, but are not limited to, calcium carbonate, calcium phosphate,various sugars, starches, cellulose derivatives, gelatin, and polymerssuch as polyethylene glycols.

Many of the compounds of the invention may be provided as salts withpharmaceutically compatible counterions. Pharmaceutically compatiblesalts may be formed with many acids, including but, not limited to,hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc.Salts tend to be more soluble in aqueous or other protonic solvents thanare the corresponding free base forms.

Suitable routes of administration may, for example, include oral,rectal, transmucosal, transdermal, or intestinal administration; orparenteral delivery, including intramuscular, subcutaneous,intramedullary injections, as well as intrathecal, directintraventricular, intravenous, intraperitoneal, intranasal, orintraocular injections.

Alternately, one may administer the compound in a local rather thansystemic manner, for example, via injection of the compound directlyinto an affected area, often in a depot or sustained releaseformulation.

Furthermore, one may administer the drug in a targeted drug deliverysystem, for example, in a liposome coated with an antibody specific foraffected cells. The liposomes will be targeted to and taken upselectively by the cells.

The pharmaceutical compositions generally are administered in an amounteffective for treatment or prophylaxis of a specific indication orindications. It is appreciated that optimum dosage will be determined bystandard methods for each treatment modality and indication, taking intoaccount the indication, its severity, route of administration,complicating conditions and the like. In therapy or as a prophylactic,the active agent may be administered to an individual as an injectablecomposition, for example, as a sterile aqueous dispersion, preferablyisotonic. A therapeutically effective dose further refers to that amountof the compound sufficient to result in amelioration of symptomsassociated with such disorders. Techniques for formulation andadministration of the compounds of the instant application may be foundin “Remington's Pharmaceutical Sciences,” Mack Publishing Co., Easton,Pa., latest edition. For administration to mammals, and particularlyhumans, it is expected that the daily dosage level of the active agentwill be from 0.001 mg/kg to 10 mg kg, typically around 0.01 mg/kg. Thephysician in any event will determine the actual dosage which will bemost suitable for an individual and will vary with the age, weight andresponse of the particular individual. The above dosages are exemplaryof the average case. There can, of course, be individual instances wherehigher or lower dosage ranges are merited, and such are within the scopeof this invention.

The compounds of the invention may be particularly useful in animaldisorders (veterinarian indications), and particularly mammals.

The invention further provides diagnostic and pharmaceutical packs andkits comprising one or more containers filled with one or more of theingredients of the aforementioned compositions of the invention.Associated with such container(s) can be a notice in the form prescribedby a governmental agency regulating the manufacture, use or sale ofpharmaceuticals or biological products, reflecting approval by theagency of the manufacture, use or sale of the product for humanadministration.

DEFINITIONS

To facilitate a complete understanding of the invention, the termsdefined below have the following meaning:

Agonist refers to any molecule or pharmaceutical agent, such as a drugor hormone, which enhances the activity of another molecule.

Antagonist refers to any molecule or pharmaceutical agent, such as adrug or hormone, which inhibits or extinguishes the activity of anothermolecule.

Chemical Derivative. As used herein, a molecule is said to be a“chemical derivative” of another molecule when it contains additionalchemical moieties not normally a part of the molecule. Such moieties canimprove the molecule's solubility, absorption, biological half life, andthe like. The moieties can alternatively decrease the toxicity of themolecule, eliminate or attenuate any undesirable side effect of themolecule, and the like. Moieties capable of mediating such effects aredisclosed in Mack E. W., 1990, Remington's Pharmaceutical Sciences, MackPublishing Company, Easton, Pa., 13^(th) edition. Procedures forcoupling such moieties to a molecule are well known in the art.

Compositions include genes, proteins, polynucleotides, peptides,compounds, drugs, and pharmacological agents.

Control region refers to a nucleic acid sequence capable of or requiredfor, assisting or impeding initiation, termination, or otherwiseregulating the transcription of a gene. The control region may include apromoter, enhancer, silencer and/or any other regulatory element. Acontrol region also includes a nucleic acid sequence that may or may notbe independently or exclusively sufficient to initiate, terminate, orotherwise regulate transcription, however, is capable of effecting suchregulation in association with other nucleic acid sequences.

Desaturase refers to a fatty acid desaturase, which is an enzyme capableof generating a double bond in the hydrocarbon region of a fatty acidmolecule.

Disorder as used herein refers to derangement or abnormality ofstructure or function. Disorder includes disease.

Drug. Drugs include, but are not limited to proteins, peptides,degenerate peptides, agents purified from conditioned cell medium,organic molecules, inorganic molecules, antibodies or oligonucleotides.The drug can be naturally occurring or synthetically or recombinantlyproduced.

Enhancer is a nucleic acid sequence comprising a DNA regulatory elementthat enhances or increases transcription when bound by a specifictranscription factor or factors. Moreover, an enhancer may function ineither orientation and in any location (upstream or downstream relativeto the promoter) to effect and generate increased levels of geneexpression when bound by specific factors. In addition, according to thepresent invention, an enhancer also refers to a compound (i.e. testcompound) that increases or promotes the enzymatic activity of theelongase gene, and/or increases or promotes the transcription of thegene.

Fatty Acids are a class of compounds comprising a long saturated or monoor polyunsaturated hydrocarbon chain and a terminal carboxyl group.

Fatty Acid Delta-5-Desaturase (D5D) is an enzyme capable of generating adouble bond between carbons 5 and 6 from the carboxyl group in a fattyacid molecule.

Fatty Acid Delta-6-Desaturase is an enzyme capable of generating adouble bond between carbons 6 and 7 from the carboxyl group in a fattyacid molecule.

Fatty Acid Elongase is an enzyme required for the addition of an acetylgroup or a 2-carbon chain to the carboxyl end of a fatty acid.

Functional Enzyme, as used herein, refers to a biologically active ornon-active protein with a known enzymatic activity.

Functional Derivative. A “functional derivative” of a sequence, eitherprotein or nucleic acid, is a molecule that possesses a biologicalactivity (either functional or structural) that is substantially similarto a biological activity of the protein or nucleic acid sequence. Afunctional derivative of a protein can contain post-translationalmodifications such as covalently linked carbohydrate, depending on thenecessity of such modifications for the performance of a specificfunction. The term “functional derivative” is intended to include the“fragments,” “sequences,” “variants,” “analogs,” or “chemicalderivatives” of a molecule.

Gene refers to a nucleic acid molecule or a portion thereof, thesequence of which includes information required for the production of aparticular protein or polypeptide chain. The polypeptide can be encodedby a full-length sequence or any portion of the coding sequence, so longas the functional activity of the protein is retained. A gene maycomprise regions preceding and following the coding region as well asintervening sequences (introns) between individual coding sequences(exons). A “heterologous” region of a nucleic acid construct (i.e. aheterologous gene) is an identifiable segment of DNA within a largernucleic acid construct that is not found in association with the othergenetic components of the construct in nature. Thus, when theheterologous gene encodes a mammalian elongase gene, the gene willusually be flanked by a promoter that does not flank the structuralgenomic DNA in the genome of the source organism.

Host system may comprise a cell, tissue, organ, organism or any partthereof, which provides an environment or conditions that allow for, orenable, transcription and/or transcription. Identity, similarity,homology or homologous, refer to relationships between two or morepolynucleotide sequences, as determined by comparing the sequences. Inthe art, identity also means the degree of sequence relatedness betweenpolynucleotide sequences, as the case may be, as determined by the matchbetween strings of such sequences. Both identity and similarity can bereadily calculated (Lesk A. M., ed., 1988, Computational MolecularBiology, Oxford University Press, NY; Smith D. W., ed., 1993,Biocomputing. Informatics and Genome Project, Academic Press, NY;Griffin A. M. and Griffin H. G., eds., 1994, Computer Analysis ofSequence Data, Part 1, Humana Press, New Jersey; von Heijne G., 1987,Sequence Analysis in Molecular Biology, Academic Press, NY and GribskovM. and Devereux J., eds., 1991, Sequence Analysis Primer, M StocktonPress, NY). While there exist a number of methods to measure identityand similarity between two polynucleotide sequences, both terms are wellknown to skilled artisans (von Heijne G., 1987, Sequence Analysis inMolecular Biology, Academic Press, NY; Cribskov M. and Devereux J.,eds., 1991, Sequence Analysis Primer, M Stockton Press, NY and CarilloH. and Lipman D., 1988, SIAM J. Applied Math., 48: 1073). Methodscommonly employed to determine identity or similarity between sequencesinclude, but are not limited to those disclosed in Carillo H. and LipmanD., 1988, SIAM J Applied Math., 48: 1073. Methods to determine identityand similarity are codified in computer programs. Computer programmethods to determine identity and similarity between two sequencesinclude, but are not limited to, GCC program package (Devereux et al.,1984, Nucl. Acid Res., 12: 387-395), BLASTP, BLASTN and FASTA (Altschulet al., 1990, J. Molec. Biol., 215: 403-410).

Isolated means altered “by the hand of man” from its natural state;i.e., that, if it occurs in nature, it has been changed or removed fromits original environment, or both. For example, a naturally occurringpolynucleotide naturally present in a living organism in its naturalstate is not “isolated,” but the same polynucleotide separated fromcoexisting materials of its natural state is “isolated”, as the term isemployed herein. As part of or following isolation, such polynucleotidescan be joined to other polynucleotides, such as DNA, for mutagenesis, toform fusion proteins, and for propagation or expression in a host, forinstance. The isolated polynucleotides, alone or joined to otherpolynucleotides such as vectors, can be introduced into host cells, inculture or in whole organisms. Introduced into host cells in culture orin whole organisms, such DNA still would be isolated, as the term isused herein, because they would not be in their naturally occurring formor environment. Similarly, the polynucleotides may occur in acomposition, such as a media formulations, solutions for introduction ofpolynucleotides, for example, into cells, compositions or solutions forchemical or enzymatic reactions, for instance, which are not naturallyoccurring compositions, and, therein remain isolated polynucleotideswithin the meaning of that term as it is employed herein.

Mutation. A “mutation” is any detectable change in the genetic material.A mutation can be any (or a combination of) detectable, unnatural changeaffecting the chemical or physical constitution, mutability,replication, phenotypic function, or recombination of one or moredeoxyribonucleotides; nucleotides can be added, deleted, substitutedfor, inverted, or transposed to new positions with and withoutinversion. Mutations can occur spontaneously and can be inducedexperimentally by application of mutagens or by site-directedmutagenesis. A mutant polypeptide can result from a mutant nucleic acidmolecule.

Nucleic acid construct refers to any genetic element, including, but notlimited to, plasmids and vectors, that incorporate polynucleotidesequences. For example, a nucleic acid construct may be a vectorcomprising a promoter or control region that is operably linked to aheterologous gene.

Operably linked as used herein indicates the association of a promoteror control region of a nucleic acid construct with a heterologous genesuch that the presence or modulation of the promoter or control regioninfluences the transcription of the heterologous gene, including genesfor reporter sequences. Operably linked sequences may also include twosegments that are transcribed onto the same RNA transcript. Thus, twosequences, such as a promoter and a “reporter sequence” are operablylinked if transcription commencing in the promoter produces an RNAtranscript of the reporter sequence.

Plasmids. Starting plasmids disclosed herein are either commerciallyavailable, publicly available, or can be constructed from availableplasmids by routine application of well known, published procedures.Many plasmids and other cloning and expression vectors that can be usedin accordance with the present invention are well known and readilyavailable to those of skill in the art. Moreover, those of skill readilymay construct any number of other plasmids suitable for use in theinvention.

Polynucleotides(s) of the present invention may be in the form of RNA,such as mRNA, or in the form of DNA, including, for instance, cDNA andgenomic DNA obtained by cloning or produced by chemical synthetictechniques or by a combination thereof. The DNA may be double-strandedor single-stranded. Single-stranded polynucleotides may be the codingstrand, also known as the sense strand, or it may be the non-codingstrand, also referred to as the anti-sense strand. Polynucleotidesgenerally refers to any polyribonucleotide or polydeoxyribonucleotide,which may be unmodified RNA or DNA or modified RNA or DNA. Thus, forinstance, polynucleotides as used herein refers to, among others,single- and double-stranded DNA, DNA that is a mixture of single- anddouble-stranded regions or single-, double- and triple-stranded regions,single- and double-stranded RNA, and RNA that is mixture of single- anddouble-stranded regions, hybrid molecules comprising DNA and RNA thatmay be single-stranded or, more typically, double-stranded, ortriple-stranded, or a mixture of single- and double-stranded regions. Inaddition, polynucleotide as used herein refers to triple-strandedregions comprising RNA or DNA or both RNA and DNA. The strands in suchregions may be from the same molecule or from different molecules. Theregions may include all of one or more of the molecules, but moretypically involve only a region of some of the molecules. One of themolecules of a triple-helical region often is an oligonucleotide. Asused herein, the term polynucleotide also includes DNA or DNA thatcontain one or more modified bases. Thus, DNA or DNA with backbonesmodified for stability or for other reasons are “polynucleotides” asthat term is intended herein. Moreover. DNA or DNA comprising unusualbases, such as inosine, or modified bases, such as tritylated bases, toname just two examples, are polynucleotides as the term is used herein.It will be appreciated that a great variety of modifications have beenmade to DNA and RNA that serve many useful purposes known to those ofskill in the art. The term polynucleotide as it is employed hereinembraces such chemically enzymatically or metabolically modified formsof polynucleotides, as well as the chemical forms of DNA and RNAcharacteristic of viruses and cells, including simple and complex cells,inter alia. Polynucleotides embraces short polynucleotides oftenreferred to as oligonucleotide(s). It will also be appreciated that RNAmade by transcription of this doubled stranded nucleotide sequence, andan antisense strand of a nucleic acid molecule of the invention or anoligonucleotide fragment of the nucleic acid molecule, are contemplatedwithin the scope of the invention. An antisense sequence is constructedby inverting the sequence of a nucleic acid molecule of the invention,relative to its normal presentation for transcription. Preferably, anantisense sequence is constructed by inverting a region preceding theinitiation codon or an unconserved region. The antisense sequences maybe constructed using chemical synthesis and enzymatic ligation reactionsusing procedures known in the art.

Promoter. Refers to a nucleic acid sequence comprising a DNA regulatoryelement capable of binding RNA polymerase directly or indirectly toinitiate transcription of a downstream (3′ direction) gene. Inaccordance with the present invention, a promoter of a nucleic acidconstruct that includes a nucleotide sequence, wherein the nucleotidesequence may be linked to a heterologous gene such that the induction ofthe promoter influences the transcription of the heterologous gene.

Purified. A “purified” protein or nucleic acid is a protein or nucleicacid preparation that is generally free of contaminants, whetherproduced recombinantly, chemically synthesized or purified from anatural source.

Recombinant refers to recombined or new combinations of nucleic acidsequences, genes, or fragments thereof which are produced by recombinantDNA techniques and are distinct from a naturally occurring nucleic acidsequence.

Regulatory element refers to a deoxyribonucleotide sequence comprisingthe whole, or a portion of, a nucleic acid sequence to which anactivated transcriptional regulatory protein, or a complex comprisingone or more activated transcriptional regulatory proteins, binds so asto transcriptionally modulate the expression of an associated gene orgenes, including heterologous genes.

Reporter gene is a nucleic acid coding sequence whose product is apolypeptide or protein that, is not otherwise produced by the host cellor host system, or which is produced in minimal or negligible amounts inthe host cell or host system, and which is detectable by various knownmethods such that the reporter gene product may be quantitativelyassayed to analyse the level of transcriptional activity in a host cellor host system. Examples include genes for luciferase, chloramphenicolacetyl transferase (CAT), beta-galactosidase, secreted placentalalkaline phosphatase and other secreted enzymes.

Silencer refers to a nucleic acid sequence or segment of a DNA controlregion such that the presence of the silencer sequence in the region ofa target gene suppresses the transcription of the target gene at thepromoter through its actions as a discrete DNA segment or through theactions of trans-acting factors that bind to these genetic elements andconsequently effect a negative control on the expression of a targetgene.

Stringent hybridization conditions are those which are stringent enoughto provide specificity, reduce the number of mismatches and yet aresufficiently flexible to allow formation of stable hybrids at anacceptable rate. Such conditions are known to those skilled in the artand are described, for example, in Sambrook et al., 1989, MolecularCloning, 2nd Edition, Cold Spring Harbor Laboratory Press, Cold SpringHarbour, N.Y. or Ausubel et al., 1994-, Current Protocols in MolecularBiology, John Wiley & Sons, NY. By way of example only, stringenthybridization with short nucleotides may be carried out at 5-10° C.below the T_(M) using high concentrations of probe such as 0.01-1.0pmole/ml. Preferably, the term “stringent conditions” meanshybridization will occur only if there is at least 95% and preferably atleast 97% identity between the sequences.

Tag refers to a specific short amino acid sequence, or theoligonucleotide sequence that encodes it, wherein said amino acid ornucleic acid sequence may comprise or encode, for example, a c-mycepitope and/or a string of six histidine residues recognizable bycommercially available antibodies. In practice, a tag facilitates thesubsequent identification and purification of a tagged protein.

Tagged protein as used herein refers to a protein comprising a linkedtag sequence. For example, a tagged protein includes a mammalianelongase polypeptide linked to a c-myc epitope and six histidineresidues at the carboxyl terminus of the amino acid sequence.

Test compounds as used herein encompass small molecules (e.g. smallorganic molecules), pharmacological compounds or agents, peptides,proteins, antibodies or antibody fragments, and nucleic acid sequences,including DNA and RNA sequences.

Transfection refers to a process whereby exogenous or heterologous DNA(i.e. a nucleic acid construct) is introduced into a recipienteukaryotic host cell. Therefore, in eukaryotic cells, the acquisition ofexogenous DNA into a host cell is referred to as transfection. Inprokaryotes and eukaryotes (for example, yeast and mammalian cells)introduced DNA may be maintained on an episomal element such as aplasmid or integrated into the host genome. With respect to eukaryoticcells, a stably transfected cell is one in which the introduced DNA hasbecome integrated into a chromosome so that it is inherited by daughtercells through chromosome replication. This stability is demonstrated bythe ability of the eukaryotic cell to establish cell lines or clonescomprised of a population of daughter cells containing the introducedDNA.

Transformation refers to a process whereby exogenous or heterologous DNA(i.e. a nucleic acid construct) is introduced into a recipientprokaryotic host cell. Therefore, in prokaryotic cells, the acquisitionof exogenous DNA into a host cell is referred to as transformation.Transformation in eukaryotes refers to the conversion or transformationof eukaryotic cells to a state of unrestrained growth in culture,resembling a tumorigenic condition. In prokaryotes and eukaryotes (forexample, yeast and mammalian cells) introduced DNA may be maintained onan episomal element such as a plasmid or integrated into the hostgenome. With prokaryotic cells, a stably transformed bacterial cell isone in which the introduced DNA has become integrated into a chromosomeso that it is inherited by daughter cells through chromosomereplication. This stability is demonstrated by the ability of theprokaryotic cell to establish cell lines or clones comprised of apopulation of daughter cells containing the introduced DNA.

Transfection/transformation as used herein refers to a process wherebyexogenous or heterologous DNA (e.g. a nucleic acid construct) has beenintroduced into a eukaryotic or prokaryotic host cell or into a hostsystem.

Variant(s) of polynucleotides are polynucleotides that differ innucleotide sequence from another, reference polynucleotide. A “variant”of a protein or nucleic acid is meant to refer to a moleculesubstantially similar in structure and biological activity to either theprotein or nucleic acid. Thus, provided that two molecules possess acommon activity and can substitute for each other, they are consideredvariants as that term is used herein even if the composition orsecondary, tertiary, or quaternary structure of one of the molecules isnot identical to that found in the other, or if the amino acid ornucleotide sequence is not identical. Generally, differences are limitedso that the nucleotide sequences of the reference and the variant areclosely similar overall and, in many regions, identical. Changes in thenucleotide sequence of the variant may be silent. That is, they may notalter the amino acids encoded by the polynucleotide. Where alterationsare limited to silent changes of this type a variant will encode apolypeptide or polynucleotide with the same amino acid sequence as thereference. Changes in the nucleotide sequence of the variant may alterthe amino acid sequence of a polypeptide encoded by the referencepolynucleotide. Such nucleotide changes may result in amino acidsubstitutions, additions, deletions, fusions and truncations in thepolypeptide or polynucleotide encoded by the reference sequence.

Vector. A plasmid or phage DNA or other DNA sequence into which DNA canbe inserted to be cloned. The vector can replicate autonomously in ahost cell, and can be further characterized by one or a small number ofendonuclease recognition sites at which such DNA sequences can be cut ina determinable fashion and into which DNA can be inserted. The vectorcan further contain a marker suitable for use in the identification ofcells transformed with the vector. Markers, for example, aretetracycline resistance or ampicillin resistance. The words “cloningvehicle” are sometimes used for “vector.”

The terminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims. Unlessdefined otherwise, all technical and scientific terms used herein havethe same meanings as commonly understood by one of ordinary skill in theart to which this invention belongs.

The present invention is further described and will be better understoodby referring to the working examples set forth below. These non-limitingexamples are to be considered illustrative only of the principles of theinvention. Since numerous modifications and changes will readily occurto those skilled in the art, it is not desired to limit the invention tothe exact construction and operation shown and described. Accordingly,all suitable modifications and equivalents may be used and will fallwithin the scope of the invention and the appended claims.

EXAMPLES

The present invention is further described by the following examples.These examples, while illustrating certain specific aspects of theinvention, do not portray the limitations or circumscribe the scope ofthe disclosed invention.

Example 1 Cloning ELG1

ELG1 was cloned into the pYES2/CT yeast expression vector (Invitrogen)using PCR. Two plasmid constructions were made for the production of theELG1 protein with either a C-terminal tag containing the V-5 epitope andpolyhistidine peptide (ELG1/V5-His), or the ELG1 protein without the tag(ELG1). The forward primer (5′-CACGCGGGTACCAGGATGGAGGCTGTTGTGAAC-3′)(SEQ. ID. NO. 14) contains the translation start codon and a KpnI site(underlined). The reverse primers for cloning ELG1 and ELG1/V5-His.5′-ATATCACGATGCGGCCGCTCAGTTGGCCTTGACCTTGGC-3′ (SEQ. ID. NO. 15) and5′-ATATCACGATGCGGCCGCCAGTTGGCCTTGACCTTGGC-3′ (SEQ. ID. NO. 16),respectively, contain a NotI site (underlined). The reverse primer forcloning ELG1 provides the translation stop codon. The reverse primer forcloning ELG1/V5-His only contains 2 of the 3 bases of the stop codon,therefore, placing the gene in frame with the tag provided by thevector.

PCR was carried out using Advantage-HF polymerase (Clontech) as per themanufacturer's instructions. The SuperScript human leukocyte cDNAlibrary (Gibco BRL) was used as the DNA template for cloning ELG1,pTh1009.1 (defined below) was used as the template for cloningELG1/V5-His.

The PCR products were gel purified, digested with KpnI and NotI, andligated into pYES2/CT cut with the same enzymes. The ligation productswere used to transform E. coli strain INVαF′ (Invitrogen). Plasmids wereisolated and their inserts were sequenced. Plasmids coding for ELG1 andELG1/V5-His were designated pTh1009.1 (FIG. 7) and pTh1009.2 (FIG. 18),respectively.

Example 2 Cloning ELG2 Obtaining Complete Coding Sequence for ELG2

Clones containing the complete coding sequence for ELG2 were obtainedfrom the SuperScript human leukocyte cDNA library (Gibco BRL) using theGeneTrapper cDNA Positive Selection System (Gibco BRL) as per themanufacturer's instructions. The sequence of the oligonucleotide used toprobe the library and repair the captured cDNA target was5′-GTAACAGGAGTATGGGAAGGCA-3′ (SEQ. ID. NO. 17). The repaired DNA wasused to transform UltraMax DH5α-FT cells (Gibco BRL). Clones containingELG2 were identified by colony PCR using 5′-TTGGACTCACACTGCTGTCTCT-3′(SEQ. ID. NO. 18) and 5′-GTGTGGCACCAAAATAAGAGTG-3′ (SEQ. ID. NO. 19) asgene specific primers and Platinum Taq DNA polymerase (Gibco BRL).Plasmid DNA was isolated from selected colonies and their inserts weresequenced. The nucleotide sequence obtained was used to identify theopen reading frame for ELG2 and to design primers for cloning ELG2 intoa yeast expression vector. A plasmid containing the complete ELG2 codingsequence was designated pSh1010.1.

Cloning ELG2 into Expression Vector

ELG2 was cloned into the pYES2/CT yeast expression vector (Invitrogen)using PCR. Two plasmid constructions were made for the production of theELG2 protein with either a C-terminal tag containing the V-5 epitope andpolyhistidine peptide (ELG2/V5-His), or the ELG2 protein without the tag(ELG2). The forward primer (5′-CACGCGGGATCCCAAATGGAACATTTGATGCATCAC-3)(SEQ. ID. NO. 20) contains the translation start codon and a BamHI site(underlined). The reverse primers for cloning ELG2 and ELG2/V5-His,5′-ATATCACGATGCGGCCGCTCAATCCTTCCGCAGCTTCC-3′ (SEQ. ID. NO. 21) and5′-ATATCACGATGCGGCCGCCAATCCTTCCGCAGCTTCC-3′ (SEQ. ID. NO. 22),respectively, contain a NotI site (underlined). The reverse primer forcloning ELG2 provides the translation stop codon. The reverse primer forcloning ELG2/V5-His only contains 2 of the 3 bases of the stop codon,therefore, placing the gene in frame with the tag provided by thevector.

PCR was carried out using Advantage-HF polymerase (Clontech) as per themanufacturer's instructions. pSh 1010.1 was used as the DNA template forcloning ELG2. pMr1014.1 (described below) was used as the DNA templatefor ELG2/V5-His.

The PCR products were gel purified, digested with BamHI and NotI, andligated into pYES2/CT cut with the same enzymes. The ligation productswere used to transform E. coli strain TOP10F′ (Invitrogen). Plasmidswere isolated and their inserts were sequenced. Plasmids coding for ELG2and ELG2/V5-His were designated pTh1014.1 and pTh1014.2, respectively.

Example 3 Cloning ELG3

ELG3 was cloned into the pYES2/CT yeast expression vector (Invitrogen)using PCR. Two plasmid constructions were made for the production of theELG3 protein with either a C-terminal tag containing the V-5 epitope andpolyhistidine peptide (ELG3/V5-His), or the ELG3 protein without the tag(ELG3). The forward primer (5′-CACGCGGGATCCATCATGGAACATCTAAAGGCC-3′)(SEQ. ID. NO. 23) contains the translation start codon and a BamHI site(underlined). The reverse primers for cloning ELG3 and ELG3/V5-His,5′-ATATCACGATGCGGCCGCTTATTGTGCTTTCTTGTTCATCACTCC-3′ (SEQ. ID. NO. 24)and 5′-ATATCACGATGCGGCCGCTTTTGTGCTTTCTTGTTCATCACTCC-3 (SEQ. ID. NO. 25),respectively, contain a NotI site (underlined). The reverse primer forcloning ELG3 provides the translation stop codon. The reverse primer forcloning ELG3/V5-His only contains 2 of the 3 bases of the stop codon,therefore, placing the gene in frame with the tag provided by thevector.

PCR was carried out using Advantage-BH polymerase (Clontech) as per themanufacturer's instructions. cDNA prepared from ZR-75-1 cells (ATCC No.CRL-1500) was used as the DNA template. This cDNA was prepared byisolating RNA from the ZR-75-1 cells using Trizol reagent (Gibco BRL) asper the manufacturer's instructions and then reverse transcribing theRNA using MuLV reverse transcriptase and random hexamers as describedfor the GeneAmp RNA PCR kit (PE Applied Biosystems).

PCR products were gel purified, digested with BamHI and voti, andligated into pYES2/CT cut with the same enzymes. The ligation productswere used to transform E. coli strain TOP10F′ (Invitrogen). Plasmidswere isolated and their inserts were sequenced. Plasmids coding for ELG3and ELG3/V5-His were designated pTh1015.1 and pTh1017.1, respectively.

ELG3 was also cloned into the pBEVY-L yeast expression vector (Miller etal., 1998, Nucl. Acids Res., 26: 3577-3583) under the control of theconstitutive glyceraldehyde 3-phosphate dehydrogenase promoter. The ELG3coding sequence was obtained by restricting pTh1015.1 with BamHI andXbaI, and gel purifying the 0.9 kb fragment. The pBEVY vector wasrestricted with BamHI and EcoRI, or XbaI and EcoRI, and the ˜1 kb and ˜6kb fragments, respectively, were gel purified. The three fragments wereligated and the ligation products were used to transform E. Coli strainINVαF′ (Invitrogen). A plasmid containing the ELG3 gene was isolated andidentified by restriction analysis. The insert DNA was confirmed by DNAsequencing and the plasmid designated pLh5015.1 (FIG. 19).

Example 4 Cloning ELG4 Obtaining Complete Coding Sequence for ELG4

A cDNA clone with an incomplete coding sequence for ELG4 was obtainedfrom the SuperScript human leukocyte cDNA library (Gibco BRL) using theGeneTrapper cDNA Positive Selection System (Gibco BRL) as per themanufacturer's instructions. The sequence of the oligonucleotide used toprobe the library and repair the captured cDNA target was5′-GCCAGCCTACCAGAAGTATTTG-3′ (SEQ. ID. NO. 26). The repaired DNA wasused to transform UltraMax DH5α-FT cells (Gibco BRL). A clone containingELG4 was identified by colony PCR using 5′-GCGCAAGAAAAATAGCCAAG-3′ (SEQ.ID. NO. 27) and 5′-AATGATGCACGCAAAGACTG-3′ (SEQ. ID. NO. 28) as genespecific primers and Platinum Taq DNA polymerase (Gibco BRL). PlasmidDNA was isolated and the insert was sequenced. The plasmid wasdesignated pSh1026.1. The complete coding sequence for ELG4 could not bedetermined, however, an open reading frame containing the C-terminus ofthe ELG4 protein was identified. Subsequent cloning (described below)revealed that pSh1026.1 contains an ELG4 variant with an internaldeletion of nucleotides 210-255 of the coding sequence.

The nucleotide sequence obtained from pSh1026.1 was used to design aforward (5′-CACGCGGGATCCCTGATGAATACAGAGCCGTGG-3′) (SEQ. ID. NO. 29) andreverse (5′-ATATCACGATGCGGCCGCTCAATTATCTTTGTTTTTGCAAGTTCC-3′) (SEQ. ID.NO. 30) primer for cloning ELG4 by PCR. These primers contain a BamHIand NotI site, respectively (underlined). The forward primer includesthe first possible translation start codon identified in pSh 1026:1. Thereverse primer provides the translation stop codon. PCR was carried outusing Advantage HF polymerase (Clontech) as per the manufacturer'sinstructions. The SuperScript human leukocyte cDNA library (Gibco BRL)was used as the DNA template.

The PCR products were gel purified, digested with BamHI and NotI, andligated into pYES2/CT (Invitrogen) cut with the same enzymes. Theligation products were used to transform E. coli strain TOP10(Invitrogen). Plasmids were isolated and their inserts were sequenced. Aplasmid containing the complete coding sequence for ELG4 as well as 108nucleotides of 5′-UTR was designated pTh1030.1.

Cloning ELG4 into Expression Vector

ELG4 was cloned into the pYES2/CT yeast expression vector using PCR. Twoplasmid constructions were made for the production of the ELG4 proteinwith either a C-terminal tag containing the V-5 epitope andpolyhistidine peptide (ELG4N5-Vis), or the ELG4 protein without the tag(ELG4). The forward primer (5′-CACGCGGGATCCCTGATGCAAAAGCCCATTAATATTC-3′)(SEQ. ID. NO. 31) contains the translation start codon and a BamHI site(underlined). The reverse primers for cloning ELG4 and ELG4/V5-His,5′-ATATCACGATGCGGCCGCTCAATTATCTTTGTTTTTGCAAGTTCC-3′, (SEQ. ID. NO. 32)and 5′-ATATCACGATGCGGCCGCCAATTATCT TTTTTGCAAGTTCC-3′, (SEQ. ID. NO. 33)respectively, contain a NotI site (underlined). The reverse primer forcloning ELG4 provides the translation stop codon. The reverse primer forcloning ELG4/V5-His only contains 2 of the 3 bases of the stop codon,therefore, placing the gene in frame with the tag provided by thevector.

PCR was carried out using Advantage-HF polymerase (Clontech) as per themanufacturer's instructions. pTh1030.1 was used as the DNA template forELG4 and pTh1021.1 (described below) was used as the template forELG4V5-His.

The PCR products were gel purified, digested with BamHI and NotI, andligated into pYES2/CT cut with the same enzymes. The ligation productswere used to transform E. coli strain TOP10 (Invitrogen). Plasmids wereisolated and their inserts were sequenced. Plasmids coding for ELG4 andELG4V5-His were designated pTh1021.1 and pTh1021.2, respectively.

Example 5 Cloning ELG5

ELG5 was cloned into the pYES2/CT yeast expression vector (Invitrogen)using PCR. Two plasmid constructions were made for the production of theELG5 protein with either a C-terminal tag containing the V-5 epitope andpolyhistidine peptide (ELG5V5-His), or the ELG5 protein without the tag(ELG5). The forward primer(5′-CACGCGGGATCCAAAATGAACATGTCAGTGTTGACTTTACAAG-3′) (SEQ. ID. NO. 34)contains the translation start codon and a BamHI site (underlined). Thereverse primers for cloning ELG5 and ELG5V5-His,5′-ATATCACGATGCGGCCGCCTATTCAGCTTTCGTTGTTTTCCTC-3′ (SEQ. ID. NO. 35) and5′-ATATCACGATGCGGCCGCCATTCAGTTTCGTTGTTTCCTC-3′, (SEQ. ID. NO. 36)respectively, contain a NotI site (underlined). The reverse primer forcloning ELG5 provides the translation stop codon. The reverse primer forcloning ELG5/V5-His only contains 2 of the 3 bases of the stop codon,therefore, placing the gene in frame with the tag provided by thevector.

PCR was carried out using Advantage-HF polymerase (Clontech) as per themanufacturer's instructions. The ProQuest human liver cDNA library(Gibco BRL) was used as the DNA template.

The PCR products were gel purified, digested with BamHI and NotI, andligated into pYES2/CT cut with the same enzymes. The ligation productswere used to transformed E. coli strain TOP10 (Invitrogen). Plasmidswere isolated and their inserts were sequenced. Plasmids coding for ELG5and ELG5V5-His were designated pTh1018.1 and pTh1019.1, respectively.

Example 6 Cloning ELG6

ELG6 was cloned into the pYES2/CT yeast expression vector (Invitrogen)using PCR. Two plasmid constructions were made for the production of theELG6 protein with either a C-terminal tag containing the V-5 epitope andpolyhistidine peptide (ELG6V5-His), or the ELG6 protein without the tag(ELG6). The forward primer(5′-CACGCGGGATCCAAAAATGGTCACAGCCATGAATGTCTC-3′) (SEQ. ID. NO. 37)contains the translation start codon and a BamHI site (underlined). Thereverse primers for cloning ELG6 and ELG6/V5-His,5′-ATATCACGATGCGGCCGCTCACTGGCTCTTGGTCTTGGC-3′ (SEQ. ID. NO. 38) and5′-ATATCACGATGCGGCCGCCACTGGCTCTTGGTCTTGGC-3′(SEQ. ID. NO. 39),respectively, contain a NotI site (underlined). The reverse primer forcloning ELG6 provides the translation stop codon. The reverse primer forcloning ELG6V5-His only contains 2 of the 3 bases of the stop codon,therefore, placing the gene in frame with the tag provided by thevector.

PCR was carried out using Advantage-HF polymerase (Clontech) as per themanufacturer's instructions. The SuperScript human leukocyte cDNAlibrary (Gibco BRL) was used as the DNA template.

The PCR products were gel purified, digested with BamHI and NotI, andligated into pYES2/CT cut with the same enzymes. The ligation productswere used to transform E. coli strain TOP10 (Invitrogen). Plasmids wereisolated and their inserts were sequenced. Plasmids coding for ELG6 andELG6V5-His were designated pTh1041.1 and pTh1042.1, respectively.

Example 7 Cloning ELG7

ELG7 was cloned into the pYES2/CT yeast expression vector (Invitrogen)using PCR. Two plasmid constructions were made for the production of theELG7 protein with either a C-terminal tag containing the V-5 epitope andpolyhistidine peptide (ELG7V5-His), or the ELG7 protein without the tag(ELG7). The forward primer (5′-CACGCGGGATCCAAAATGGGGCTCCTGGACTCGGAGC-3′) (SEQ. ID. NO. 40) contains the translationstart codon and a BamHI site (underlined). The reverse primers forcloning ELG7 and ELG7V5-His.5′-ATATCACGATGCGGCCGCTTAATCTCCTTTTGCTTTTCCATTTTTCTGC-3′ (SEQ. ID. NO.41) and 5′-ATATCACGATGCGGCCGCTTATCTCCTTTTGCTTTTCCATTTTTCTGC-3 ′ (SEQ.ID. NO. 42), respectively, contain a NotI site (underlined). The reverseprimer for cloning ELG7 provides the translation stop codon. The reverseprimer for cloning ELG7V5-His only contains 2 of the 3 bases of the stopcodon, therefore, placing the gene in frame with the tag provided by thevector.

PCR was carried out using Platinum Taq DNA polymerase (Gibco BRL) as perthe manufacturer's instructions. The SuperScript human leukocyte cDNAlibrary (Gibco BRL) was used as the DNA template.

The PCR products were gel purified, digested with BamHI and NotI, andligated into pYES2/CT cut with the same enzymes. The ligation productswere used to transform E. coli strain TOP10 (Invitrogen). Plasmids wereisolated and their inserts were sequenced. Plasmids coding for ELG7 andELG7V5-His were designated pTh1044.1 and pTh1045.1, respectively.

Example 8 Determination of Tissue Distribution by Northern Blot Analysis

A membrane containing poly(A)⁺ RNA from 12 different human tissues(brain, heart, skeletal muscle, colon, thymus, spleen, kidney, liver,small intestine, placenta, lung and peripheral blood leukocytes) waspurchased from Clontech (Human 12-lane MTN blot). Northern blot analysiswas carried out using standard procedures (Ausubel et al., 1994-,Current Protocols in Molecular Biology, John Wiley & Sons, NY). Thehybridization solution contained 10% dextran sulphate. Probes wereprepared by labelling cDNA using [alpha-³²P]dCTP and Rediprime II RandomPrime Labelling System (Amersham Pharmacia Biotech). The cDNA probes forELG1, ELG3, ELG5, and ELG6 corresponded to the complete CDS for thegenes. The cDNA probes for ELG2, ELG4 and ELG7 corresponded to bases209-514, 408-726 and 113-566 of the CDS, respectively. The membrane waswashed at high stringency using 0.25×SSC, 0.1% SDS at 55° C.

Example 9 Cloning Human ELG1 Control Region

The ELG1 control region (989 bp) is cloned from human leukocyte genomicDNA by PCR. The control region is amplified by PCR using syntheticforward and reverse primers starting at positions −2865 by and −1877 byupstream from the translation initiation codon, ATG. The forward andreverse primers used for cloning human ELG1 control region by PCRamplification are 5′-GGAAGATCTTACAGGCTCGTGAGGCTTCCCTCCCG-3′ (SEQ. ID.NO. 43) and 5′-GGAAGATCTCCGGCAGGAGGGACCAAGGCT-3′ (SEQ. ID. NO. 44),respectively. The BglII recognition sequence (underlined) is included tofacilitate cloning. The PCR amplification is performed in a Perkin-ElmerGeneAMP PCR system 9700 instrument. For example, the PCR is performed ina 50 μl reaction volume containing 0.5 μg of genomic DNA, 0.4 μM of eachprimer, 1× dNTP mix (Clontech, California), 1× cDNA PCR reaction buffer(Clontech) and 1× Advantage cDNA polymerase mix (Clontech).

The conditions for the PCR reaction are:

-   -   7 cycles at 94° C. for 2 seconds, 72° C. for 3 minutes    -   32 cycles at 94° C. for 2 seconds, 67° C. for 3 minutes    -   67° C. for 4 minutes

The PCR product is gel-purified using QIAquick gel extraction kit(Qiagen, Germany) and ligated into the TA cloning vector pCR11(Invitrogen) according to manufacturers instruction. The ligationproduct is used to transform E. coli TOP10 strain (Invitrogen). Theresulting plasmids are screened by restriction analysis and confirmed byDNA sequencing. The human ELG1 control region is then recloned from thepCR11/ELG1 control region construct into the luciferase reporter vectorpGL3-Basic (Promega). The resulting human ELG1 control region/reporterconstruct is used to transfect different mammalian cell lines, andreporter activity measured.

Example 10 Cloning Human ELG2 Control Region

The ELG2 control region (509 bp) is cloned from human leukocyte genomicDNA by PCR. The control region is amplified by PCR using syntheticforward and reverse primers starting at positions −53626 bp and −53118bp upstream from the translation initiation site, ATG. The forward andreverse primers used for cloning human ELG2 control region by PCRamplification are 5′-GGAAGATCTCGAGGGTGGGCTTCTGCCACCC-3′ (SEQ. ID. NO.45) and 5′-GGAAGATCTCTTTTAGCCCAAGGGGCGGCAGC-3′ (SEQ. ID. NO. 46),respectively. The BglII recognition sequence (underlined) is included tofacilitate cloning. The PCR amplification and cloning are performed asdescribed in Example 9.

The resulting human ELG2 control region/reporter construct is used totransfect different mammalian cell lines, and reporter activitymeasured.

Example 11 Cloning of the Human ELG3 Control Region

The human ELG3 control region was cloned from human leukocyte genomicDNA by nested PCR. Blood was obtained from volunteers in the presentinventors' laboratory and used to prepare genomic DNA that served astemplate. In the first PCR reaction, synthetic forward and reverseprimers starting at position −2025 bp and −1 bp, respectively, upstreamfrom the translation initiation codon, ATG of the ELG3 gene were used.The forward and reverse primers were5′-GGAAGATCTTTCGTGTGAATTTCCTTCAACTCTC-3′ (SEQ. ID. NO. 47) and5′-GGAAGATCTTGATCCGCAGCGGCTGTG-3′ (SEQ. ID. NO. 48), respectively. TheBglII recognition sequence (underlined) was included to facilitatecloning.

The PCR amplification was conducted in a Perkin-Elmer GeneAMP PCR system9700 instrument, in a 50 μl reaction volume containing 0.5 μg of genomicDNA, 0.4 μM of each primer, 1×dNTP mix (Clontech, California), 1×cDNAPCR reaction buffer (Clontech) and 1× Advantage cDNA polymerase mix(Clontech).

The conditions for the PCR reaction were:

-   -   7 cycles at 94° C. for 2 seconds, 72° C. for 3 minutes    -   32 cycles at 94° C. for 2 seconds, 67° C. for 3 minutes    -   67° C. for 4 minutes

Analysis of the PCR product by agarose gel electrophoresis revealed thatat least two primer specific bands of about 2 kb were amplified. Thisresult necessitated the use of the PCR products as a template and a newset of internal primers in a second PCR reaction to generate a uniqueprimer specific band corresponding to the ELG3 control region. Theinternal forward and reverse primers start at positions −1381 and −37respectively, upstream from the translation initiation codon, ATG. Theinternal forward and reverse primers used were5′-GGAAGATCTCCGCTACCTACAGTTACTCACTCTGC-3′ (SEQ. ID. NO. 49) and5′-GGAAGATCTGGCGATGCGCTGTCCAGGCTA-3′ (SEQ. ID. NO. 50).

The conditions for PCR reaction described herein were used for thesecond PCR reaction except for the following modifications: the secondtemperature cycle was lowered from 32 to 22 cycles, Taq DNA polymerasewas substituted for cDNA polymerase and Q solution (Qiagen) was usedaccording to manufacturer's instruction.

The PCR product was gel-purified using QiAquick gel extraction kit(Qiagen). The purified PCR product and the reporter vector pCL3-Basicwere separately digested with BglII restriction enzyme to generatecompatible ends suitable for in-frame ligation of the PCR product to theluciferase gene of pGL3-basic. The ligation product was used totransform E. coli TOP10 strain (Invitrogen). The resulting plasmid,pGh3020.1 (FIG. 20), was screened by restriction analysis and confirmedby DNA sequencing. The resulting human ELG3 control region/reporterconstruct is used to transfect different mammalian cell lines, andreporter activity measured.

Example 12 Cloning Human ELG4 Control Region

The ELG4 control region (2456 bp) is cloned from human leukocyte genomicDNA by PCR. The control region is amplified by PCR using syntheticforward and reverse primers. The forward and reverse primers used forcloning human ELG4 control region by PCR amplification are5′-CGACGCGTTGCGCCTGGCTGAACACTAC-3′ (SEQ. ID. NO. 51) and5′-GGAAGATCTCTGGGACAAACAACAGGC-3′ (SEQ. ID. NO. 52), respectively. TheMluI and BglII recognition sequences (underlined), respectively, areincluded to facilitate cloning.

The PCR amplification and cloning are performed as described in Example9.

The resulting human ELG4 control region/reporter construct is used andto transfect different mammalian cell lines, and reporter activitymeasured.

Example 13 Cloning Human ELG5 Control Region

The ELG5 control region (1411 bp) is cloned from human leukocyte genomicDNA by PCR. The control region is amplified by PCR using syntheticforward and reverse primers starting at positions −1411 bp and −1 bpupstream the translation initiation codon, ATG. The forward and reverseprimers used for cloning human ELG5 control region by PCR amplificationare 5′-CCGCTCGAGGTGAGCCACCACCGCGGCC-3′ (SEQ. ID. NO. 53) and5′-CCGCTCGAGTGGGGCTGATCTTCGGAGTCGC-3′ (SEQ. ID. NO. 54), respectively.The XhoI recognition sequence (underlined) is included to facilitatecloning.

The PCR amplification and cloning are performed as described in Example9. The resulting human ELG5 control region/reporter construct is used totransfect different mammalian cell lines, and reporter activitymeasured.

Example 14 Cloning Human ELG6 Control Region

The ELG6 control region (1937 bp) is cloned from human leukocyte genomicDNA by PCR. The control region is amplified by PCR using syntheticforward and reverse primers starting at positions −1937 bp and −1 bpupstream the initiation codon, ATG. The forward and reverse primers usedfor cloning human ELG6 control region by PCR amplification are5′-CCGAGCTCGATTAGCTGTCAGGCTATATATGGAGCC-3′ (SEQ. ID. NO. 55) and5′-CCGAGCTCCTAGTTTGCAGAAGGTCCAAAGC-3′ (SEQ. ID. NO. 56), respectively.The SacI recognition sequence (underlined) is included to facilitatecloning.

The PCR amplification and cloning are performed as described in Example9.

The resulting human ELG6 control region/reporter construct is used totransfect different mammalian cell lines, and reporter activitymeasured.

Example 15 Cloning Human ELG7 Control Region

The ELG7 control region (2000 bp) is cloned from human leukocyte genomicDNA by PCR. The control region is amplified by PCR using syntheticforward and reverse primers starting at positions −2000 bp and −1 bpupstream the translation initiation codon, ATG. The forward and reverseprimers used for cloning human ELG7 control region by PCR amplificationare 5′-CCGAGCTCGGAAATACCTGAAGCTGTTTTAAC-3′ (SEQ. ID. NO. 57) and5′-CCGAGCTCCGCGGCGATGAGCGGGC-3′ (SEQ. ID. NO. 58), respectively. TheSacI recognition sequence (underlined) is included to facilitatecloning.

The PCR amplification and cloning are performed as described in Example9.

The resulting human ELG7 control region/reporter construct is used totransfect different mammalian cell lines, and reporter activitymeasured.

Example 16 Drug Screening Assay Using ELG3 Control Region

Plasmid pGh3020.1 (FIG. 20), containing the ELG3 control region, is usedto screen test compounds that modulate the ELG3 promoter activity.Transient transfections are performed to evaluate the functionality ofthe ELG3 control region using techniques known by persons skilled in theart.

Alternatively, HepG2 cells are stably transfected with 10 μg ofpGh3020.1 and 1 μg of vector pRSV-NEO (ATCC), using 10 μl ofLipofectamine 2000 Reagent (Gibco BRL) in a 60 mm tissue culture dish asdescribed by the manufacturer. After a 24 h incubation, the cells arepassaged into two 150 mm tissue culture dishes at a 1:2 dilution andgrown for another 24 h. Geneticin (Gibco BRL) is added to the medium ata concentration of 800 μg ml. After 3-4 weeks of growth under theselection pressure of the antibiotic, the resistant clones are isolatedand characterized for their luciferase activity.

Drug screening is performed using the Luciferase Enzyme Assay System(Promega), following the manufacturer's recommendations. Briefly,transfected cells grown in a 96 well plate are exposed to testcompounds. After an appropriate incubation time, the cells are washedwith Mg²⁺ and Ca²⁺ free PBS. Cells are lysed with 20 μl of 1× LuciferaseCell Culture Lysis Reagent (CCLR, Promega). The plate is placed into aluminometer with an automatic injector. For each well, the injector adds100 μl of Luciferase Assay Reagent (Promega), and the light emissiongenerated by the reaction is read for 10 seconds after a 2 second delay.Cell cultures without a test compound are used as controls. Anysignificant difference in the luciferase activity indicates that thetest compound is modulating the ELG3 promoter activity.

This assay or other reporter assays are suitable for drug screeningusing the control region of any elongase gene.

Example 17 Drug Screening Assays Using Yeast One-Hybrid Systems

Methods for yeast one-hybrid assays are known by persons skilled in theart (Fields S, and Song O., 1989, Nature, 340: 245-246 and Ulmasov etal., 1997, Science, 276: 1865-1868). Reagents and/or kits arecommercially available for the assays, e.g., the Matchmaker One-HybridSystem (Clontech).

This assay is suitable for all of the elongase control regions describedherein.

The known target elements, or elongase control region ‘bait’ is insertedupstream of a reporter gene (e.g. HIS3) and integrated into the yeastgenome to make a new reporter strain. The yeast strain is transformedwith an activation domain (AD) fusion library to screen for DNA bindingproteins that interact with the bait DNA sequence. Binding of anAD/DNA-binding domain (DBD) hybrid protein to the target sequenceresults in activation of the reporter gene transcription and subsequentselection. For example, expression of HIS3 will allow colony growth onminimal medium lacking histidine. The cDNA encoding DNA binding protein(DBP) is isolated and characterized. The interaction is reconstructed invitro or in vivo for screening test compounds by exposing the targetelements or elongase control region to the DBP in the presence of testcompounds. The effect of the test compound is evaluated through assays,well known to those skilled in the art, that measure DNA/protein bindinginteractions.

Example 18 Drug Screening Assays Using Yeast Two-Hybrid Systems

Methods for the yeast two-hybrid assays are known by persons skilled inthe art (Fields S, and Song O., 1989, Nature, 340: 245-246 and FuruyamaK. and Sassa S., 2000, J. Clin. Invest., 105: 757-764). Reagents and/orkits are commercially available for the assays, e.g., the Hybrid HunterYeast Two-Hybrid (Invitrogen), the Matchmaker Two-Hybrid Systems(Clontech) and the HybriZAP Two Hybrid System (Stratagene).

This assay is suitable for all of the elongase genes disclosed herein.

Two physically distinct functional domains are necessary: a DNA bindingdomain (DBD) and an activation domain (AD). The elongase polypeptide ofinterest is cloned into a “bait” vector, and expressed as a hybridprotein with a DBD. A library of cDNAs encoding potential interactingproteins is cloned in frame with AD in the “prey” vector. The bait andprey vector fusion constructs are transformed into one of severalengineered yeast strains. If an interaction between bait and prey hybridproteins occurs, the AD of the prey is brought into close contact withthe DBD and transcription of the reporter genes is activated. Positiveinteracting proteins are easily identified by plating on nutrientdeficient medium, and screening for reporter activity.

The interaction between these two proteins is reconstructed in vitro orin vivo for screening test compounds by exposing the two interactingproteins to test compounds. The effect of the test compound is evaluatedthrough assays, well known to those skilled in the art, that measureprotein/protein binding interactions.

Example 19 Functional Analysis of Human Elongases in Saccharamycescerevisiae

The example presented herein demonstrates that the human elongase genes,ELG1, ELG2, ELG3, ELG4, ELG5, ELG6 and ELG7 cloned by the inventors,encode enzymes able to elongate, by at least two carbons, n-3 and/or n-6fatty acid substrates.

Materials

Lithium [1-¹⁴C]18:3n-6, [1-¹⁴C]18:3n-3, [1-¹⁴C]20:4n-6, and[1-¹⁴C]20:5n-3 (99% radiochemical purity; specific activity: 48 to 58μCi/μmol), were purchased from NEN (Boston, Mass.). All unsaturatedfatty acids were saponified with 0.1 M LiOH and dissolved in a syntheticminimal medium lacking uracil (SC-U) with 1% tergitol.

Fatty acid free bovine serum albumin, tergitol, Tris-HCl, carbohydrates,amino acids and fatty acids were obtained from Sigma-Aldrich Canada (ON,Canada). Yeast nitrogen base without amino acids was purchased fromDifco (Becton Dickinson). All organic solvents (HPLC grade) wereobtained from Fisher-Scientific (Fair Lawn, N.J.).

Yeast Transformation

Saccharomyces cerevisiae strain INVSc1 (Invitrogen) was transformed withthe elongase constructs previously described (Examples 1-7) or pYES2/CTusing the lithium acetate method as supplied by Invitrogen. For theexpression of ELG1, ELG2, ELG3, ELG4, ELG5, ELG6 or ELG7 the yeast weretransformed with pTh1009.1, pTh1014.1, pTh1015.1, pTh1021.1, pTh1018.1,pTh1041.1 or pTh1044.1, respectively. For the expression of ELG1V5-His,ELG2V5-His, ELG3V5-His, ELG4V5-His, ELG5V5-His, ELG6/V5-His orELG7V5-His the yeast were transformed with pTh1009.2, pTh1014.2,pTh1017.1, pTh1021.2, pTh1019.1, pTh1042.1 or pTh1045.1, respectively.Recombinant yeast cells were selected on SC-U medium.

Incubation

Transformed yeast (approximately 3.2×10⁶ cells/ml; O.D.₆₀₀ 0.4) wereincubated in a 125 ml Erlenmeyer containing 10 ml of SC-U medium with 1%raffinose, 1% tergitol and 25 μM of the lithium salts of either[1-¹⁴C]18:3n-3 (1 μCi), [1-¹⁴C]18:3n-6 (1 μCi), [1-¹⁴C]20:4n-6 (2 μCi),or [1-¹⁴C]20:5n-3 (2 μCi). After 4 h incubation in an orbital incubatorat 270 rpm and 30° C., cells reached the log phase and the transgeneexpression was induced with galactose (2% final concentration). Theyeast were incubated for an additional 19 h and then harvested bycentrifugation at 5000×g for 10 minutes at 4° C.

Cells were washed with Tris-HCl buffer (100 mM, pH 8.0) containing 0.1%BSA and total lipids were extracted as described below. Theradioactivity from aliquots of the incubation medium, supernatant andcells was determined by liquid scintillation counting using aLS6500-Scintillation System (Beckman).

The host yeast transformed with pYES2/CT was used as negative control.

Lipid Extraction

Total lipids were extracted from cells with chloroform/methanol (2:1v/v) according to the method of Folch et al., 1957, J. Biol. Chem., 226:497-509. Alternatively, cells were resuspended in 1.5 ml of water andsaponified with 2 ml of 10% KOH in ethanol. The total lipid extracts orthe free fatty acids from the saponified samples were methylated usingboron trifluoride in methanol at 90° C. for 30 min. The resultant methylesters (FAME) were analyzed as described below.

Reverse Phase-High Performance Liquid Chromatography (RP-HPLC) Analysis

Analyses of radiolabelled FAME were carried out on a Hewlett Packard1090, series II chromatograph equipped with a diode array detector setat 205 nm, a radioisotope detector (model 171, Beckman, CA) with a solidscintillation cartridge (97% efficiency for ¹⁴C-detection) and areverse-phase ODS (C-18) Beckman column (250 mm×4.6 mm i.d.; 5). μmparticle size) attached to a pre-column with a μBondapak C-18 (Beckman)insert. FAME were separated isocratically with acetonitrile/water (95:5v/v) at a flow rate of 1 ml/min and were identified by comparison withauthentic standards. Alternatively, the eluted FAME were collected andthe solvent evaporated. FAME were re-dissolved in hexane for furtheranalysis by gas chromatography.

Gas Chromatography (GC) Analysis

The FAME profile was determined using a Hewlett Packard GasChromatograph equipped with an interfaced ChemStation, aflame-ionization detector and a 30 nm×0.25 μm i.d. fused silica column(HP-wax, cross linked polyethylene glycol, film thickness 0.25 pm) andHe as gas carrier. The temperatures of the injector and detector weremaintained at 225° C. and 250° C., respectively. After an initial holdof 1 min at 180° C., the column temperature was increased by 4° C./minto 190° C. (7 min hold), then by 10° C./min to 200° C. (5 min hold) andfinally by 25° C./min to 215° C. This temperature was maintained for17.9 min. FAME were identified by comparison with authentic standards.

Results

RP-HPLC analyses revealed that the exogenously added radiolabelledpolyunsaturated fatty acids were elongated by at least two carbons inyeast transformed with human elongase genes (Table 3). In yeastexpressing ELG4, 18:3n-6 was converted into 20:3n-6 which was thenelongated to 22:3n-6, 20:4n-6 was converted into 22:4n-6 which wasfurther elongated to 24:4n-6 and 18:3n-3 was converted into 20:3n-3 and22:3n-3 (FIG. 21). Yeast transformed with pYES2/CT did not elongate anyof these substrates (FIG. 22).

In yeast expressing elongases with V5-His tag, the percent elongation ofselected substrates was similar to that detected in yeast withnon-tagged enzymes (Table 4).

CONCLUSION

The functional analysis of the human ELG1, ELG2, ELG3, ELG4, ELG5, ELG6and ELG7 genes confirmed that each gene encodes a fatty acid elongasewhich is active on various PUFAs.

TABLE 3 Percent Elongation of PUFA Substrates to their Products in YeastExpressing Human Elongases 18:3n-6 20:4n-6 18:3n-3 20:5n-3 Gene Plasmid20:3 22:3 22:4 24:4 20:3 22:3 22:5 24:5 ELG1 pTh1009.1 2 nd 6 2 1 nd 2nd ELG2 pTh1014.1 62 3 39 1 16 nd 59 nd ELG3 pTh1015.1 10 nd 11 21 2 nd16 29 ELG4 pTh1021.1 20 4 24 2 10 4 15 3 ELG5 pTh1018.1 3 nd nd Nd 9 nd— — ELG6 pTh1041.1 2 nd nd nd 3 nd nd nd ELG7 pTh1044.1 nd nd nd nd 5 ndnd rnd nd: not detected —: not tested

TABLE 4 Percent Elongation of PUFA Substrates to their Products in YeastExpressing V5-His Tagged Human Elongases 18:3n-6 20:4n-6 18:3n-3 20:5n-3Gene Plasmid 20:3 22:3 22:4 24:4 20:3 22:3 22:5 24:5 ELG1 pTh1009.2 — —7 nd — — — — ELG2 pTh1014.2 73 11 — — — — — — ELG3 pTh1017.1 — — 8 15 —— — — ELG4 pTh1021.2 — — 12 nd — — — — ELG5 pTh1019.1 −5 — — — — — — —ELG6 pTh1042.1 nd nd Nd nd 3 nd nd nd ELG7 pTh1045.1 nd nd Nd nd 4 nd ndnd nd: not detected —: not tested

Example 20 Drug Screening Assay for Elongases Using Yeast

This example provides a methodology suitable for screening testcompounds that modulate the activity of recombinant elongases in wholecells and spheroplasts of Saccharomyces cerevisiae. The test compounduptake is likely to be enhanced in yeast spheroplasts due to their lackof a cell wall. Thus, this is the model of choice for assessing theeffect of low concentrations of test compounds on elongase activity.

Spheroplast Preparation

Saccharomyces cerevisiae heterologous for any of the human elongasegenes are grown in SC-U medium with 1% raffinose and 2% galactose toinduce the expression of the transgene. After 16 h incubation, cells arecentrifuged at 2060×g for 5 min at 4° C., washed once with distilledwater and centrifuged again. The volume and weight of the cell pelletare measured. Cells are suspended (1:2 w/v) in 0.1 M Tris.SO₄ (pH 9.4),10 mM DTIT and incubated at 30° C.

After 10 min incubation, the cell pellet is obtained by centrifugation,washed once (1:20 w/v) with 1.2 M sorbitol and suspended (1:1 w/v) in1.2 M sorbitol, 20 mM phosphate buffer (pH 7.4) as described elsewhere(Daum et al., 1982, J. Biol. Chem., 257: 13028-13033). A 15,800×g (1min) supernatant of lyticase is added to the cell suspension at aconcentration of 2000 U/ml and the suspension incubated at 30° C. with50 rpm shaking. Conversion to spheroplasts is checked after 40 minincubation by diluting the suspension with distilled water followed byobservation under the microscope (Schatz G. and Kovac L., 1974, Meth.Enzymol., 31A: 627-632). After 70 min incubation, approximately 90% ofthe cells are converted to spheroplasts.

Incubation of Spheroplasts with Test Compounds

Spheroplasts are harvested by centrifugation at 2060×g for 5 min at 4°C. and washed once with 1.2 M sorbitol. Spheroplasts are resuspended inSC-U medium with 1% raffinose, 1% tergitol, 1.2 M sorbitol and 2%galactose to maintain the induction conditions and to give an O.D.₆₀₀reading of approximately 2.5-3.0. A 10 ml aliquot of the spheroplastsuspension is transferred to a 125 ml Erlenmeyer flask and incubatedwith 200 μl of a test compound in ethanol (e.g. pebulate sulphoxide witha final concentration ranging from 0.01 to 100 μm) at 30° C. in anorbital incubator at 270 rpm. After 30 min incubation, 1 μCi of aselected elongase substrate (i.e., lithium salts of [1-¹⁴C]18:3n-6,[1-¹⁴C]20:4n-6, [1-¹⁴C]20:5n-3 or [1-¹⁴C]18:3n-3) is added to theculture to a final concentration of 2 to 200 μM and further incubatedfor 120 min. Cell density is determined (O.D.₆₀₀) and spheroplasts areharvested by centrifugation and washed with Tris-HCl buffer (100 mM, pH8.0) containing 0.1% BSA. Total lipids are extracted and analyzed asdescribed in Example 19.

Incubation of Whole Yeast with Test Compounds

Saccharomyces cerevisiae heterologous for any of the human elongasegenes are incubated in a 125 ml Erlenmeyer flask containing 9 ml of SC-Umedium with 1% raffinose, 1% tergitol (O.D.₆₀₀ 0.4, approximately3.2×10⁶ cells/ml) and 200 μl of a test compound in ethanol (e.g.pebulate sulphoxide, with a final concentration in the culture thatrange between 0.1 and 5 μm). After 1 h incubation in an orbitalincubator at 270 rpm and 30° C., 1 μCi of a selected elongase substrate(i.e., lithium salts of [1-¹⁴C]18:3n-6, [1-¹⁴C]20:4n-6, [1-¹⁴C]20:5n-3or [1-¹⁴]18:3n-3) is added to the culture to a final concentration of 2to 200 μM. After 4 h incubation with the inhibitor, cells reach the logphase and the transgene expression is induced with the addition of 1 mlof galactose to a final concentration of 2%. The yeast are incubated foran additional 19 h and then harvested by centrifugation at 5000×g for 10minutes at 4° C. Cells are washed with Tris-HCl buffer (100 mM, pH 8.0)containing 0.1% BSA and total lipids are extracted and analyzed asdescribed in Example 19.

Calculations

The elongase activity is determined by measuring the conversion ofradiolabelled 18:3n-6 to 20:3n-6 and 22:3n-6, 20:4n-6 to 22:4n-6 and24:4n-6, 18:3n-3 to 20:3n-3 and 22:3n-3 or 20:5n-3 to 22:5n-3 and24:5n-3. The percent inhibition is calculated as described elsewhere(Kawashima et al., 1996, Biosci. Biotech. Biochem., 60: 1672-1676):

% Inhibition=100 (activity without the inhibitor−activity with theinhibitor)/activity without the inhibitor

Example 21 Drug Screening Assay for Elongase Using Yeast Microsomes

This example teaches that microsomes from yeast with elongase transgenescontain all the enzymes required for testing the effect of testcompounds on the activity of a specific recombinant fatty acid elongase.

Materials

A sulphoxide derivative of S-propylbutylethylthiocarbamate (pebulatesulphoxide) was obtained from Zeneca Agrochemicals, UK, and dissolved inethanol at a concentration of 5 mM.

Yeast Microsome Preparation

A 51 culture of Saccharomyces cerevisiae transformed with pTh 1017.1encoding ELC3V5-His was started with a cell density of approximately3.2×10⁵ cells/ml (O.D.₆₀₀ 0.4) using SC-U medium with 1% raffinose.After 8 h of incubation at 30° C. in an orbital shaker at 270 rpm,galactose was added to a final concentration of 2%. Yeast were incubatedfor an additional 12 h until they were harvested by centrifugation at2060×g for 10 minutes at 4° C. and washed with water. The cell pelletwas resuspended in ⅓ of its volume in a pH 7.2 isolation buffer (80 mMHepes-KOH, 10 mM KCl, 320 mM sucrose, 2 mM PMSF and a protease inhibitorcocktail). The cell suspension was poured into a mortar containingliquid N2 and ground with sand using a ceramic pestle. The yeast powderwas transferred to a conical test tube, to which ⅔ of the pellet volumeof isolation buffer was added. The sand was removed by centrifugation at228×g for 1 min and the suspension centrifuged at 10,000×g for 20 min toseparate cell debris, nuclei and mitochondria. The supernatant wascentrifuged at 106,000×g for 1.5 h to obtain the microsomal pellet,which was resuspended in storage buffer (80 mM Hepes-KOH, 10 mM KCl, 320mM sucrose, 1 mM PMSF and a protease inhibitor cocktail) to a finalprotein concentration of 20 μg/μl. The protein concentration wasmeasured by the method of Lowry et al. (1951, J. Biol. Chem., 193:265-275) with bovine serum albumin as standard.

Incubation of Yeast Microsomes with Pebulate Sulphoxide

The activity of ELG3V5-His was determined by measuring the conversion of[1-¹⁴C]20:5n-3 to [1-¹⁴C]22:5n-3 and [1-¹⁴C]24:5n-3. Reactions werestarted by adding 500 μg of yeast microsomal protein to pre-incubatedtubes containing 0.20 μCi of the substrate fatty acid at a finalconcentration of 7.2 μM in 0.25 ml of 80 mM Hepes-KOH (pH 7.2) with 43mM MgCl₂, 1.0 mM ATP, 500 μM NADPH, 100 μM coenzyme A, 100 μMmalonyl-CoA (as lithium salt) and pebulate sulphoxide at concentrationsthat ranged between 1 to 100 μM. The tubes were vortexed vigorously andafter 30 min incubation at 37° C. in a shaking water bath, the reactionswere stopped by the addition of 2 ml of 10% (w/v) KOH in ethanol. Lipidsin the incubation mixture were saponified at 80° C. for 45 min under N₂.The samples were then left in ice for 5 min before acidification with750 μl of concentrated HCl. The fatty acids were extracted with hexaneand esterified with BF₃ in methanol at 90° C. for 30 min. The fatty acidmethyl esters were analyzed by HPLC as described in Example 19.

Results

The enzyme activity was expressed in percent conversion of radiolabelled20:5n-3 into its elongation products. Alternatively, it can be expressedin pmol of the fatty acids produced/mg microsomal protein/min.

Table 5 shows the effect of a thiocarbamate derivative (pebulatesulphoxide) on the ELG3V5-His activity when 20:5n-3 was provided assubstrate. Pebulate sulphoxide at 100 μM substantially reducedelongation, by approximately 27%. This effect was mainly due to areduction in the synthesis of 22:5n-3 rather than in the production ofits metabolite, 24:5n-3.

TABLE 5 Effect of Pebulate Sulphoxide on the Elongation of[1-¹⁴C]20:5n-3 in Microsomes of Yeast Expressing ELG3/V5-His. Pebulatesulphoxide % conversion [μM] 22:5n-3 24:5n-3 Total 0 13.7 5.0 18.7 113.8 5.6 19.4 10 12.8 6.6 19.4 50 11.3 4.6 15.9 100 9.4 4.3 13.7 Valuesexpressed are the average (dispersions 10%) of two determinations.

Example 22 Isolation of Recombinant Elongases from Yeast

This example provides a methodology for the isolation of recombinantelongase from yeast homogenate or microsomes. The purified enzyme isuseful for drug screening or for antibody production.

Yeast Homogenate and Microsome Preparation

Yeast cell fractionation was performed as described in Example 21 usingyeast expressing ELG3/V5-His.

Elongase Solubilization

Yeast cell homogenate or yeast microsomes were resuspended insolubilization buffer (80 mM HEPES-KOH pH 7.2, 10 mM KCl, 320 mMsucrose, 1 mM PMSF, protease inhibitor cocktail, and 0.5 M NaCl) at 1.3or 4 mg/ml, respectively. Zwittergent 3-14, n-octyl-beta-glucopyranosideor n-octyl-beta-thioglucopyranoside (Calbiochem, CA) was added to afinal concentration of 2%, with a detergent:protein ratio of 15:1. Themixture was incubated for 2 h at 4° C. with stirring and thencentrifuged at 106,000×g for 1 h. The supernatant was removed and storedat −80° C. until use. The pellet was resuspended in ¼ volume of thesupernatant using solubilization buffer. The efficiency of eachdetergent to solubilize the elongase was determined by Western blotanalysis as described below.

SDS-PAGE and Western Blot Analysis

Supernant (60 μl) or pellet suspension (20 μl) was mixed with 15 μl or 5μl of 5× sample loading buffer (1× concentration: 50 mM Tris-HCl pH 8.0,2% SDS, 10 mM beta-mercaptoethanol, 0.1% bromophenol blue, 10%glycerol), respectively, and boiled at 100° C. for 5 minutes. Molecularweight standards (Santa Cruz Biotechnology, CA), controls, 25 μl of thesupernatant, and 12.5 μl of the pellet were loaded on 12% pre-castSDS-polyacrylamide gels. After electrophoresis, the protein waselectro-transferred onto a PVDF membrane (Bio-Rad). The membrane wasincubated with a blocking solution and subsequently probed with ananti-V5-HRP antibody as recommended by the manufacturer (Invitrogen).The membrane was washed and the antibody was detected using the enhancedchemiluminescence reagent, ECL (Amersham-Pharmacia Biotech.). Themembrane was exposed to autoradiography film (Labscientific, NewJersey).

Zwittergent 3-14 was the most effective detergent in solubilizingELG3/V5-His, the majority of the tagged protein having been detected inthe 106,000×g supernatant.

Immobilized Metal Ion Affinity Chromatography (IMAC)

The supernatant containing the solubilized enzyme is loaded onto apre-equilibrated HiTrap chelating (Ni²⁺ charged iminodiacetate) column(Pharmacia) attached to a fast protein liquid chromatography system(Pharmacia). The column is washed with 50 mM sodium phosphate pH 8.0.The tagged protein is eluted with the same buffer containing imidazoleranging from 0 to 500 mM and further concentrated by ultrafiltrationusing Centriprep (Amicon) concentrators.

Alternatively, Macro-Prep ceramic hydroxyapatite (Bio-Rad, CA), TALONmetal affinity resin (a Cobalt-based IMAC resin, Clontech, California),Ni-nitriloacetic acid resin (Novagen, Wis.) or other similar resin isused.

Example 23 Drug Screening Assay for Elongase Using Purified Enzyme

The concentrated enzyme (Example 22) is incubated at 30-37° C. in 0.25ml of 80 mM Hepes-KOH (pH 7.2) with 6 mM egg phosphatidylcholine, 2%Triton X-100, 0.4% sodium deoxycholate, 43 mM MgCl₂, 1.0 mM ATP, 500 μMNADPH, 10 μM coenzyme A, 100 μM malonyl-CoA (as lithium salt), 0.20 μCiof the substrate fatty acid (i.e., radiolabelled eicosapentaenoyl-CoA)at a final concentration of 7.2 μM and a test compound (e.g., pebulatesulphoxide) at concentrations ranging between 0.01 to 100 μM. The tubesare vortexed vigorously and after 30 min incubation at 37° C. in ashaking water bath the reactions are stopped by the addition of 2 ml of10% (w/v) KOH in ethanol.

Total lipids are extracted and methyl ester analyzed as described inExample 19.

Example 24 Validation of Drug Screening Assays Described in Examples 20,21 and 23 Using Rat Liver Microsomes Preparation of Rat Liver Microsomes

Wistar rats under light halothane (15% in mineral oil) anesthesia weresacrificed by exsanguination during periods of high enzyme activity.Livers were immediately rinsed with cold 0.9% NaCl solution, weighed andminced with scissors. All procedures were performed at 4° C. unlessspecified otherwise. Livers were homogenized in a solution (1:3 w/v)containing 0.25 M sucrose, 62 mM potassium phosphate buffer (pH 7.0),0.15 M KCl, 1.5 mM N-acetylcysteine, 5 mM MgCl₂, and 0.1 mM EDTA using 4strokes of a Potter-Elvehjem tissue homogenizer. The homogenate wascentrifuged at 10,400×g for 20 min to pellet mitochondria and cellulardebris. The supernatant was filtered through a 3-layer cheesecloth andcentrifuged at 105,000×g for 60 min. The microsomal pellet was gentlyresuspended in the same homogenization solution with a smallglass/teflon homogenizer and stored at −80° C. The absence ofmitochondrial contamination was enzymatically assessed as describedelsewhere (Kilberg, M. S. and Christensen H. N., 1979, Biochemistry, 18:1525-1530). The protein concentration was measured by the method ofLowry et al (1951, J. Biol. Chem., 193: 265-275) with bovine serumalbumin as standard.

Incubation of Rat Liver Microsomes with Test Compounds

Reactions were performed using 500 μg of rat liver microsomal proteinwith the same concentrations of pebulate sulphoxide, radiolabelled fattyacid, conditions and procedures described in Example 21.

Results

The enzyme activity was expressed in percent conversion of radiolabelled20:5n-3 into its elongation and final delta-6-desaturation products(i.e., 22:5n-3, 24:5n-3 and 24:6n-3). When the incubation was performedunder nitrogen, the desaturation reaction did not occur. Table 6 showsthe effect of a thiocarbamate derivative (pebulate sulphoxide) on therat liver elongase activity when 20:5n-3 was provided as substrate.Pebulate sulphoxide (100 μM) reduced elongation by approximately 30%.This effect was mainly due to a reduction in the synthesis of 24:5n-3rather than in the synthesis of 22:5n-3.

TABLE 6 Effect of Pebulate Sulphoxide on the Elongation of[1-¹⁴C]20:5n-3 in Rat Liver Microsomes Pebulate sulphoxide % conversion[μM] 22:5n-3 24:5n-3 24:6n-3* Total 0 11.6 39.7 9.1 60.4 1 12.5 47.5 9.669.3 10 12.5 47.2 10.9 70.7 50 12.2 48.7 7.9 68.8 100 10.2 28.0 4.5 42.7Values are expressed as the mean (dispersion ≦10%) of twodeterminations. *24:6n-3 is the product of a delta-6-desaturation of24:5n-3.

Since the rat liver microsomal and the recombinant human elongase(Example 21) activities were similarly affected by pebulate sulphoxide,it is concluded that rat liver microsomes are suitable to use in thevalidation of drug screening assays.

Example 25 Functional Characterization of Recombinant Fatty AcidElongase and Desaturase in Yeast Co-expressing ELG3 and D6D

This example shows a partial reconstitution of the n-3 and n-6polyunsaturated fatty acid biosynthetic pathway in a heterologous hostsuch as Saccharomyces cerevisiae using human fatty acid elongase anddesaturase genes.

Materials

[1-¹⁴C]18:3n-3, [1-¹⁴C]20:4n-6, [1-¹⁴C]20:5n-3 and [1-¹⁴C]18:2n-6 (99%radiochemical purity; specific activity: 51 to 56 μCi/mol) werepurchased from NEN (Boston, Mass.). Fatty acids were saponified with 0.1M LiOH and dissolved in synthetic minimal medium lacking either leucine(SC-Leu) or uracil and leucine (SC-U-Leu), containing 1% tergitol.

Yeast Transformation

Saccharomyces cerevisiae strain INVSc1 (Invitrogen) was transformedusing the lithium acetate method as supplied by Invitrogen. The codingsequence for human delta-6-desaturase (GenBank Accession No. AF126799)was previously cloned into the pYES2/CT vector for the production of theprotein with a C-terminal tag containing the V-5 epitope andpolyhistidine peptide (D6D/V5-His) as described in Canadian PatentApplication No. 2,301,158, March, 2000, Winther et al. (plasmiddesignated pTh5002.1). For the co-expression of ELG3 and D6D/V5-His, theyeast were initially transformed with pTh5002.1. Recombinant yeast cellswere selected on SC-U medium and then transformed with pLh5015.1(Example 3). Double recombinant yeast cells containing both pTh5002.1and pLh5015.1 were selected on SC-U-Leu medium. Yeast cells transformedwith pBEVY-L alone, the cloning vector for ELG3, were selected on SC-Leumedium.

Incubation

Transformed yeast cultures (approximately 3.2×10⁶ cells/ml; O.D.₆₀₀,0.4) were divided in two experimental groups. The first group wasincubated in a 125 ml Erlenmeyer flask containing 10 ml of SC-U-Leumedium with 2% raffinose, 1% tergitol and 25 μM lithium [1-¹⁴C]20:4n-6(1 μCi) Yeast of the second group were incubated in 10 ml of SC-U-Leumedium containing 1% raffinose, 2% galactose (to induce the expressionof D6D/V5-His) and 1% tergitol. Lithium salts (1 μCi) of either[1-¹⁴C]18:3n-3, [1-¹⁴C]20:4n-6, [1-¹⁴C]20:5n-3 or [1-¹⁴C]18:2n-6 wereadded to both experimental groups at a final concentration of 25 μM.After 24 h incubation in an orbital incubator at 270 rpm and 30° C.,cells were harvested by centrifugation at 5000×g for 10 minutes at 4° C.

The cell pellet was washed with Tris-HCl buffer (100 mM, pH 8.0)containing 0.1% BSA total lipids were extracted and radiolabelled fattyacids analyzed as described in Example 19. The host yeast transformedwith pBEVY-L was used as negative control.

Results

FIGS. 23 and 24 show that only elongation products of PUIA substratesfor ELG3 were detected when galactose was absent from the culture mediumsince the expression of D6D/V5-His was not induced. The constitutivelyexpressed ELG3 was able to elongate 20:4n-6 to 22:4n-6 and 24:4n-6,20:5n-3 to 22:5n-3 and 24:5n-3, and to a lesser extent 18:3n-3 to20:3n-3. These findings are consistent with those described in Example19. ELG3 did not elongate 18:2n-6.

The elongation products of PUFA substrates for ELG3 were desaturated byD6D/V5-His when galactose was added to the medium (FIG. 24). In thisregard, 24:5n-6 and 24:6n-3 were produced from 24:4n-6 and 24:5n-3,respectively.

In the presence of galactose, transformed yeast were also able todelta-6-desaturate 18:2n-6 and 18:3n-3 to 18:3n-6 and 18:4n-3,respectively. These products were then substrates of the ELG3, whichelongated them to 20:3n-6 and 20:4n-3, respectively.

Both ELG3 and D6D/V5-His seemed to be more active on n-3 than on n-6fatty acid substrates.

Yeast transgenic for the human elongase, ELG3, and a human D6D, wereable to generate polyunsaturated fatty acids of the so called “Sprecherpathway” (Sprecher H., 2000, Biochim. Biophys. Acta, 1486: 219-231). Thepresent inventors are the first to report that products of human ELG3,24:4n-6 and 24:5n-3, are substrates of a human D6D, which is also activeon 18:2n-6 and 18:3n-3.

Example 26 Functional Characterization of Recombinant Fatty AcidElongase and Desaturase in Yeast Co-expressing ELG3 and D5D)

This example expands the inventors' findings described in Example 25.The sequential elongation and desaturation of n-3 and n-6 PUFAs in aheterologous host co-expressing human fatty acid elongase and D5D genesis demonstrated.

Materials

[1-¹⁴C]18:3n-3, [1-¹⁴C]20:3n-6 and [1-¹⁴C]18:2n-6 (99% radiochemicalpurity; specific activity: 50 to 52 μCi/μmol) were purchased from NEN(Boston, Mass.). [1-¹⁴C]-Δ^(8,11,14,17) eicosatetraenoic acid, 20:4n-3,(99% radiochemical purity; specific activity: 55 μCi/μmol) was purchasedfrom ARC (St Louis, Mo.). Fatty acids were saponified with 0.1 M LiOHand dissolved in either SC-Leu or SC-U-Leu medium, containing 1%tergitol.

Yeast Transformation

Saccharomyces cerevisiae strain INV/Sc1 (Invitrogen) was transformedusing the lithium acetate method as supplied by Invitrogen. The codingsequence for human delta-5-desaturase (GenBank Accession No. AF199596)was previously cloned into the pYES2/CT vector for the production of theprotein with a C-terminal tag containing the V-5 epitope andpolyhistidine peptide (D5D/V5-His) as described in Canadian PatentApplication No. 2,301,158, March, 2000, Winther et al.(plasmiddesignated pTh5009.1). For the co-expression of ELG3 and D5D/V5-His, theyeast were initially transformed with pTh5009.1. Recombinant yeast cellswere selected on SC-U medium and then transformed with pLh5015.1(described in Example 3). Double recombinant yeast cells containing bothpTh5009.1 and pLh5015.1 were selected on SC-U-Leu medium. Yeast cellstransformed with pBEVY-L alone. the cloning vector for ELG3, wereselected on SC-Leu medium.

Incubation

Cultures of transformed yeast (approximately 3.2×10⁶ cells/ml; O.D.₆₀₀0.4) were divided in two experimental groups. In the first group, cellswere incubated in a 125 ml Erlenmeyer flask containing 10 ml of SC-U-Leumedium with 2% raffinose and 1% tergitol. In the second group, yeastwere incubated in 11 ml of SC-U-Leu medium with 1% raffinose. 2%galactose (to induce the expression of D5D/V5-His) and 1% tergitol.Lithium salts (1 μCi) of either [1-¹⁴C]18:3n-3, [1-¹⁴C]20:3n-6.[1-¹⁴C]18:2n-6, or [1-¹⁴C]20:4n-3 were added to both experimental groupsat a final concentration of 25 μM. After 24 h incubation in an orbitalincubator at 270 rpm and 30° C., cells were harvested by centrifugationat 5000×g for 10 minutes at 4° C.

The cell pellet was washed with Tris-HCl buffer (100 mM, pH 8.0)containing 0.1% BSA. total lipids were extracted and radiolabelled fattyacids were analyzed as described in Example 19.

The host yeast transformed with pBEVY-L was used as negative control.

Results

FIG. 25 shows that 20:3n-6 was desaturated to 20:4n-6, which was furtherelongated to 22:4n-6 and 24:4n-6, when the yeast co-expressed both genesin the presence of galactose. When galactose was not added to themedium, 20:3n-6 was only elongated to 22:3n-6. Similarly, D5D/V5-Hisdesaturated 20:4n-3 producing 20:5n-3, which was then elongated to22:5n-3 and 24:5n-3. The elongation of 20:4n-3 to 22:4n-3 and 24:4n-3was also detected. Under these experimental conditions, yeastco-expressing both genes was not able to elongate and further desaturate18:2n-6. D5D/V5-His was not active on 20:3n-3, the direct elongationproduct of 18:3n-3 generated by ELG3 (FIG. 26).

CONCLUSION

Yeast co-expressing ELG3 and a human D5D, both cloned by the inventors,were able to generate substrates (i.e., 24:4n-6 and 24:5n-3) of the socalled “Sprecher pathway” (Sprecher H., 2000, Biochim. Biophys. Acta,1486: 219-231).

Example 27 Drug Screening Assays Using Whole Cells, Spheroplasts orMicrosomes of Yeast Co-Expressing ELG3 and either Human D6D or D5D

The following assays are designed to identify compounds that affect thehuman elongase ELG3 and/or the human desaturases using one host systemor any part thereof.

Spheroplast and Microsome Preparation

Transformed Saccharomyces cerevisiae cells are grown in SC-U-Leu mediumwith 1% raffinose and 2% galactose to induce the expression of thedesaturase transgenes. After 16 h incubation, spheroplasts are obtainedas described in Example 20.

Microsomes from host cells expressing both elongase and desaturase genesare prepared using the liquid N₂ and differential centrifugation methodsdescribed in Example 21.

Incubation of Whole Yeast Cells, Spheroplasts or Microsomes with TestCompounds

In these assays with yeast cells containing elongase and desaturasetransgenes, the use of SC-U-Leu medium is required to maintain selectionpressure. Transformed yeast are incubated with or without galactose toassess [sic] the effect of the test component on the activity of ELG3and the desaturases or the elongase alone, respectively. The substratesof choice are 20:3n-6 or 20:5n-6 for yeast expressing ELG3 and D5D orELG3 and D6D, respectively. The incubation conditions of whole yeastcells, spheroplasts or microsomes with test compounds are the same asthose described in Examples 20 and 21. Regardless of the host systemused, the effect of the test compound on the activity of the recombinantenzymes is determined by the RP-HPLC or GC analysis of the relativeamounts of FAME produced by ELG3 and/or the desaturases as described inExample 19.

Example 28 Elongation of PUFAs in Primary Cultures of Leukocytes fromControl and STZ-Induced Diabetic Rats

The present example describes the capability of leukocytes to elongatebut not desaturate PUFAs. The example also provides details of how theelongation of 18:3n-6 and 18:2n-6 is affected in rats with STZ-induceddiabetes.

Materials

RPMI 1640 medium was obtained from Gibco BRL. Streptozotocin(2-desoxy-2-methylnitrosoamino carbonyl amino-D-glucopyranose) wassupplied by Sigma.

Animals

Female Wistar rats were obtained from Charles Rivers, St-Constant,Quebec. Animals were housed in barrier-maintained rooms at 22±2° C., atarget relative humidity of 50±10% with 15 air changes per hour and a 12h light/dark cycle. Water and regular chow were provided ad libitum.

All animals were monitored daily according to standard procedures incompliance with the Canadian Council of Animal Care guidelines foranimal experimentation. Fifteen randomly selected rats wereintraperitoneally (I.P.) injected with 50 mg of STZ per kg of bodyweight. Nine days later, animals received a second dose of STZ (75 mg/kgbody weight). A second group of 12 rats which were sham injected withsterile 0.9% NaCl served as control. Two and 7 weeks after the last I.P. injection, control and STZ-treated rats (blood glucose levels 21to >33 mmoles/l) were put under light halothane (15% in mineral oil)anesthesia and sacrificed by exsanguination. Blood was collected into a10 ml syringe containing 200 μl of a 5% solution of EDTA asanticoagulant.

Leukocyte Isolation

Leukocytes were obtained by mixing I volume of whole blood with 5volumes of sterile erythrocyte lysis buffer (Qiagen, CA). The cellsuspension was incubated for 20 min on ice and centrifuged at 400×g for10 min at 4° C. The supernatant was discarded and the leukocyte pelletwas washed and resuspended in 550 μl of 0.9% saline. Aliquots were takenfor cell counting. Cellular protein content was measured using themethod of Lowry et al (1951, J. Biol. Chem., 193: 265-275) with bovineserum albumin as standard.

Incubation

The present inventors' preliminary studies carried out with leukocytesisolated from Wistar rats showed that leukocytes can elongate 18:2n-6,18:3n-3, 18:3n-6, 20:3n-6 and 20:4n-6 with the elongation of 18:2n-6 and18:3n-6 being 6% and 66%, respectively, within 24 h. Based on theseresults and due to the impairment of D6D in diabetes, 18:2n-6 and18:3n-6, substrate and product of D6D, respectively, were selected forthe incubation of leukocytes from control and STZ-induced diabetic rats.No delta-6-desaturation on 18:2n-6, 18:3n-3 or delta-5-desaturation on20:3n-6, was detected.

Leukocytes from the 2 and 7 week control group, as well as from the 2and 7 week STZ-treated rat group, were incubated in RPMI 1640 mediumwith glutamine, 10% fetal calf serum and antibiotics (50 IU/mlpenicillin, 50 μg/ml streptomycin) with 5 μM [1-¹⁴C]18:3n-6 (0.6 μCi)for 10 min to 24 h or with 5 μM of [1-¹⁴C]18:2n-6 (0.6 μCi) for 24 h.

At the end of each incubation, the cell pellet was obtained bycentrifugation at 400×g for 10 min at 4° C. Cells were washed with PBScontaining 0.1% bovine serum albumin. Total cellular lipids wereextracted with chloroform:methanol (2:1 v/v). Fatty acids weremethylated with BF₃ and analyzed by RP-HPLC as described in Example 19.Alternatively, FAME can be analyzed by GC as described in Example 19.

Results

Table 7 shows that leukocytes from STZ-induced diabetic rats rapidlyconverted 18:3n-6 into 20:3n-6. There was a significant increase in theactivity of the elongation system in the STZ group, regardless of thetime after the last I.P. STZ injection. Conversely, there was anapproximately 50% reduction in the elongation of 18:2n-6 to 20:2n-6 inleukocytes obtained 2 weeks after the STZ injection (Table 8). Therewere no significant changes in the elongation of 18:2n-6 to 20:2n-6 inleukocytes from animals sacrificed 7 weeks after the STZ treatment.

TABLE 7 Conversion of 18:3n-6 into 20:3n-6 in Leukocytes fromSTZ-Induced Diabetic Rats Sacrificed 2 or 7 Weeks Post-InductionIncubation 2 weeks 7 weeks time (h) STZ Control STZ Control 0 0 0 0 00.16 50 ± 8 31 ± 9 37 ± 9 33 ± 4  0.5 115 ± 26  70 ± 12 112 ± 10 71 ± 151 288 ± 23 200 ± 16 190 ± 92 143 ± 31  24 nt nt 1008 ± 98  628 ± 156Values are expressed in pmol of 20:3n-6 produced/mg cellular protein andrepresent the mean ± S.D. of 6 rats. nt: not tested

TABLE 8 Conversion of 18:2n-6 into 20:2n-6 in Leukocytes fromSTZ-Induced Diabetic Rats Sacrificed 2 or 7 Weeks Post-Induction 2 weeks7 weeks STZ Control STZ Control 322 ± 119 126 ± 27 147 ± 22 128 ± 32Leukocytes were incubated for 24 h.Values are expressed in pmol of 20:2n-6 produced/mg cellular protein andrepresent the mean ±S.D. of 6 rats.

PUIA metabolism is altered in leukocytes of rats with STZ-induceddiabetes. Therefore, leukocytes are an appropriate model to assess themodification or regulation of the elongation system in disease (e.g.,diabetes).

Example 29 Elongation of PUFAs in Primary Cultures of Leukocytes fromHumans

This example shows that human leukocytes are a suitable model to assesselongase activity on 18:3n-6. This assay may be used in clinical trialsto determine alterations in the elongation system in diseases such asdiabetes.

Peripheral venous blood from fasted healthy volunteers (30 to 50 yearsof age) was obtained using 10 ml Vacutainers (Vacutainer Systems, NewJersey) containing EDTA as anticoagulant. Leukocytes were isolated usingthe techniques described in Example 28. The incubation of leukocyteswith 5 μM [1-¹⁴C]18:3n-6 (0.6 μCi) for 10 to 60 min was performed underthe same conditions described in Example 28.

Results

Table 9 demonstrates that human leukocytes have a capability to rapidlyelongate 18:3n-6 to 20:3n-6, similar to that found in rat leukocytes(Example 28). No delta-5-desaturation activity was detected on 20:3n-6.

TABLE 9 Conversion of 18:3n-6 into 20:3n-6 in Leukocytes from Male andFemale Volunteers Incubation time (h) Male Female 0 0 0 0.16 24 ± 5 25 ±4 1 142 ± 60 157 ± 50 24 1479 ± 249 2233 ± 778Values are expressed in pmol of 20:3n-6 produced/mg cellular protein andrepresent the mean ±S.D. of 4 volunteers.

REFERENCES

-   U.S. Pat. No. 3,817,837, June, 1974, Rubinstein et al.-   U.S. Pat. No. 3,850,752, November, 1974, Schuurs et al.-   U.S. Pat. No. 3,939,350, February, 1976, Kronick et al.-   U.S. Pat. No. 3,996,345, December, 1976, Ullman et al.-   U.S. Pat. No. 4,275,149, June, 1981, Litman et al.-   U.S. Pat. No. 4,277,437, July, 1981, Maggio-   U.S. Pat. No. 4,366,241, December, 1982, Tom et al.-   U.S. Pat. No. 4,399,216, August, 1983, Axel et al.-   U.S. Pat. No. 4,704,362, November, 1987, Itakura et al.-   U.S. Pat. No. 4,766,075, August, 1988, Goeddal et al.-   U.S. Pat. No. 4,784,950, November, 1988, Hagen et al.-   U.S. Pat. No. 4,801,542, January, 1989, Murray et al.-   U.S. Pat. No. 4,816,567, March, 1989, Cabilly et al.-   U.S. Pat. No. 4,935,349, June, 1990, McKnight et al.-   U.S. Pat. No. 5,130,238, July, 1992, Malek-   International Patent Application No. WO 00/55330, September, 2000,    Napier J. A.-   Patent Cooperation Treaty International Publication No. WO 00/12720,    March, 2000, Mukerji et al.-   European Published Application No. 0320308, June, 1989, Backman et    al.-   Patent Cooperation Treaty International Publication No. WO 93/05182,    March, 1993, Bruice-   International Patent Application No. WO 88/04300, June, 1988, Cech    et al.-   Canadian Patent Application No. 2,301,158, March, 2000, Winther et    al.-   Altschul et al., 1990, J. Molec. Biol., 215: 403-410-   Ausubel et al., 1994-, Current Protocols in Molecular Biology, John    Wiley & Sons, Inc., NY-   Been M. D. and Cech T. R., 1986, Cell, 47: 207-216-   Bennett et al., 1995, J. Mol. Recognit., 8: 52-58-   Carillo H. and Lipman D., 1988, SIAM J. Applied Math., 48: 1073-   Caskey C. T., 1987, Science, 236:1223-1229-   Church et al., 1988, Proc. ATatl. Acad. Sci., 81: 1991-1995-   Cinti et al., 1992, Prog. Lipid Res., 31: 1-51-   Connolly B. A., 1987, Nucl. Acids Res., 15: 3131-3139-   Copsey et al., 1988, Genetically Engineered Human Therapeutic Drugs,    Stockton Press, N.Y.-   Cotter et al., 1995, Diabetic Neuropathy. New Concepts and Insights,    Elsevier Science B. V., Amsterdam, pp. 115-120-   Cotton et al., 1985, Proc. Natl. Acad. Sci., 85: 4397-4401-   Daum et al., 1982, J. Biol. Chem., 257: 13028-13033-   Davis et al., 1986, Basic Methods in Molecular Biology, Elsevier,    N.Y.-   Deutcher, M., ed., 1990, Guide to Protein Purification, Meth. in    Enzymol., Vol. 182-   Devereux et al., 1984, Nucl. Acids Res., 12: 387-395-   Dines et al., 1993, Diabetologia, 36: 1132-1138-   Erickson et al., 1992, Ann. Rep. Med. Chem., 27: 271-289-   Fields S. and Song O., 1989, Nature, 340: 245-246-   Flavell et al., 1978, Cell, 15: 25-41-   Folch et al., 1957, J. Biol. Chem., 226: 497-509-   Furuyama K. and Sassa S., 2000, J. Clin. Invest., 105: 757-764-   Geever et al., 1981, Proc. Natl. Acad. Sci., 78: 5081-5085-   Goding J. W., 1996, Monoclonal Antibodies: Principles and Practice:    Production and Application of Monoclonal Antibodies in Cell Biology,    Biochemistry and Immunology, 3^(rd) edition, Academic Press, NY-   Goszcz et al., 1998, Methods Find. Exp. Clin. Pharmacol., 20:    439-445-   Gribskov M. and Devereux J., eds., 1991, Sequence Analysis Primer, M    Stockton Press, NY-   Griffin A. M. and Griffin H. G., eds., 1994, Computer Analysis of    Sequence Data, Part 1, Humana Press, New Jersey-   Gyuris et al., 1993, Cell, 75: 791-803-   Hamy et al., 1997, Proc. Natl. Acad. Sci., 94: 3548-3553-   Hanahan et al., 1983, J. Mol. Biol., 166: 557-580-   Harlow E. and Lane D., eds., 1988, Antibodies. A Laboratory Manual,    Cold Spring Harbor, N.Y.-   Haseloff J. and Gerlach W. L., 1988, Nature, 334: 585-591-   Horrobin D. F. (ed.), 1990, Omega-6 Essential Fatty Acids:    Pathophysiology and Roles in Clinical Medicine, Wiley-Liss, NY-   Houbenweyl et al., 1987, Methods of Organic Chemistry, Wansch E.    (ed), Vol. 15 I and II, Thieme, Germany-   Huse et al., 1989, Science, 246: 1275-1281-   Hwang et al., 1999, Proc. Natl. Acad. Sci., 96: 12997-13002-   Innis M. A. and Gelfand D. H., 1989, PCR Protocols, A Guide to    Methods and Applications,-   Innis M. A., Gelfand D. H., Shinsky J. J. and White T. J. (eds),    Academic Press, NY, pp. 3-12-   Izant J. G. and Weintraub H., 1984, Cell, 36: 1007-1015-   Jackson et al., 1990, EMBO J., 9: 3153-3162-   James et al., 1995, Plant Cell, 7: 309-319-   Johanson et al., 1995, J. Biol. Chem., 270: 9459-9471-   Kawashima et al., 1996, Biosci. Biotech. Biochem., 60: 1672-1676-   Kilberg M. S. and Christensen H. N., 1979, Biochemistry, 18:    1525-1530-   Kohler G. and Milstein C., 1975, Nature, 256: 495-497-   Kraemer et al., 1993, J. Lipid Res., 34: 663-671-   Landegren et al., 1988, Science, 241: 1077-1080-   Landegren et al., 1989, Science, 242: 229-237-   Leonard et al., 2000, Biochem J., 350: 765-770-   Lesk A. M., ed., 1988, Computational Molecular Biology, Oxford    University Press, NY-   Llewellyn et al., 1987, J. Mol. Biol., 195: 115-123-   Lowry et al., 1951, J. Biol. Chem., 193: 265-275-   Mack E. W., 1990, Remington's Pharmaceutical Sciences, Mack    Publishing Company, Easton, Pa., 13^(th) edition-   Margolskee et al., 1988, Mol. Cell. Biol, 8: 2837-2847-   Mazza G. and Domah B. D. (eds.), 2000, Herbs, Botanicals, and Teas,    Technomic Publishers, Lancaster, Pa.-   McLaughlin et al., 1988, J. Virol., 62: 1963-1973-   Mei et al., 1998, Biochemistry, 37: 14204-14212-   Merrifield, 1964, J. Am. Chem. Assoc., 85: 2149-2154-   Miller et al., 1998, Nucl. Acids Res., 26: 3577-3583-   Moss et al., 1987, Annu. Rev. Immunol, 5: 305-324-   Myers et al., 1985, Science, 230: 1242-1246-   Myers et al., 1986, Cold Spring Harbour Sym. Quant. Biol., 51:    275-284-   Nilsson T. and Warren G., 1994, Curr. Opin. Cell Biol., 6: 517-521-   Oh et al., 1997, J. Biol. Chem., 272: 17376-17384-   Okano et al., 1988, EMBO J., 7: 3407-3412-   Orkin et al., 1988, Prog. Med. Genet., 7: 130-142-   Rasmussen et al., 1987, Meth. Enzymol., 139: 642-654-   Riemersma et al., 1986, Br. Med. J. (Clin. Res. Ed.), 292: 1423-1427-   Rosenberg et al., 1985, Nature, 313: 703-706-   Saiki et al., 1985, Science, 230: 1350-1353-   Saiki, et al., 1986, Nature, 324: 163-166-   Sambrook et al., 1989, Molecular Cloning, 2^(nd) Edition, Cold    Spring Harbor Laboratory Press, Cold Spring Harbour, N.Y.-   Schatz G. and Kovac L., 1974, Meth. Enzymol., 31A: 627-632-   Shanklin et al., 1994, Biochemistry, 33: 12787-12794-   Singer et al., 1984, Prostaglandins Leukot. Med., 15: 159-165-   Smith D. W. ed., 1993, Biocomputing: Informatics and Genome Project,    Academic Press. NY-   Sonnhammer et al., 1998, In Proc. of Sixth Int. Conf. on Intelligent    Systems for Molecular Biology, AAAI Press, CA, pp. 175-182-   Sprecher H., 2000, Biochim. Biophys. Acta, 1486: 219-231-   Suneja et al., 1990, Biochim. Biophys. Acta, 1042: 81-85-   Thompson et al., 1994, Nucl. Acids Res., 22: 4673-4680-   Toke D. A. and Martin C. E., 1996, J. Biol. Chem., 271: 18413-18422-   Tvrdik et al., 2000, J. Cell. Biol., 149: 707-717-   Tvrdik et al., 1997, J. Biol. Chem., 272: 31738-31746-   Ulmasov et al., 1997, Science, 276: 1865-1868-   von Heijne C., 1987, Sequence Analysis in Molecular Biology,    Academic Press, NY-   Waldmann T. A., 1991, Science, 252: 1657-1661-   Wallace et al., 1986, Cold Spring Harbour Symp. Quant. Biol., 51:    257-261-   Zaug A. J. and Cech T. R., 1986, Science, 231: 470-475-   Zaug et al., 1984, Science, 224: 574-578-   Zaug et al., 1986, Nature, 324: 429-433

1. An isolated polynucleotide sequence, comprising a polynucleotidesequence encoding a polypeptide having elongase activity which isselected from the group consisting of: (a) a sequence comprising SEQ IDNO: 8; (b) a sequence at least 95% homologous with sequence (a); (c) asequence at least 98% homologous with sequence (a); and (d) a sequenceat least 99% homologous with sequence (a).
 2. A vector transformed withthe polynucleotide of claim
 1. 3. An isolated host cell comprising thepolynucleotide sequence of claim 1, wherein said sequence isheterologous to the host cell.
 4. An isolated host cell comprising apolynucleotide encoding a polypeptide sequence having elongase activityselected from the group consisting of: (a) a sequence comprising SEQ IDNO: 9; (b) a sequence which is at least 95% homologous with a sequenceof (a); (c) a sequence which is at least 98% homologous with a sequenceof (a); and (d) a sequence which is at least 99% homologous with asequence of (a) in a host cell which is heterologous to said sequence.